CD44

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CD44 antigen precursor (CDw44) (Epican) (Extracellular matrix receptor III) (ECMR-III) (GP90 lymphocyte homing/adhesion receptor) (HUTCH-I) (Heparan sulfate proteoglycan) (Hermes antigen) (Hyaluronate receptor) (Phagocytic glycoprotein 1) (PGP-1) (Phagocytic glycoprotein I) (PGP-I) [LHR] [MDU2] [MDU3] [MIC4]

Publications[править]

Hyaluronan goes to great length.

Hyaluronan is a major non-protein component of extracellular matrix that affects biomechanical properties of tissues and interacts with cell receptors. Hyaluronan is a linear glycosaminoglycan composed of repeating disaccharides of (β, 1-4)-glucuronic acid (GlcUA) and (β, 1-3)-N-acetyl glucosamine (GlcNAc). The length of hyaluronan can range from an oligomer to an extremely long form up to millions of daltons. The concept that emerged in the field is that high (HMW-HA) and low (LMW-HA) molecular weight hyaluronans have different biological properties and trigger different signaling cascades within the cells. LMW-HA is associated with inflammation, tissue injury and metastasis, while HMW-HA improves tissue homeostasis and has anti-inflammatory and antimetastatic properties. HMW-HA is used in the clinic to treat arthritis, and as a filler in surgery and in the form of rinses to treat local inflammation. However, HMW-HA products used in the clinic come in a range of sizes between 0.5-6 mDa that are used interchangeably. Remarkably, the tissues of a long-lived and cancer-resistant rodent, the naked mole rat, contain abundant HA of very high molecular weight. While human fibroblasts secrete HA up to 2 MDa, naked mole rat fibroblasts produce HA of 6-12 MDa. Does this very high HMW-HA (vHMW-HA) differ functionally from HMW-HA? We found that vHMW-HA has superior cytoprotective properties compared to HMW-HA, and interacts differently with the CD44 receptor leading to distinct transcriptional changes (Takasugi [i]et al.[/i] (2020), Nat Commun). These results indicate that vHMW-HA has greater therapeutic benefits than the standard HMW-HA.


Keywords

  • aging
  • hyaluronan
  • longevity
  • naked mole rat
  • very high molecular weight hyaluronan


Naked mole-rat very-high-molecular-mass hyaluronan exhibits superior cytoprotective properties.

Naked mole-rat (NMR), the longest-living rodent, produces very-high-molecular-mass hyaluronan (vHMM-HA), compared to other mammalian species. However, it is unclear if exceptional polymer length of vHMM-HA is important for longevity. Here, we show that vHMM-HA (>6.1 MDa) has superior cytoprotective properties compared to the shorter HMM-HA. It protects not only NMR cells, but also mouse and human cells from stress-induced cell-cycle arrest and cell death in a polymer length-dependent manner. The cytoprotective effect is dependent on the major HA-receptor, CD44. We find that vHMM-HA suppresses CD44 protein-protein interactions, whereas HMM-HA promotes them. As a result, vHMM-HA and HMM-HA induce opposing effects on the expression of CD44-dependent genes, which are associated with the p53 pathway. Concomitantly, vHMM-HA partially attenuates p53 and protects cells from stress in a p53-dependent manner. Our results implicate vHMM-HA in anti-aging mechanisms and suggest the potential applications of vHMM-HA for enhancing cellular stress resistance.

MeSH Terms

  • Animals
  • Apoptosis
  • Cell Cycle Checkpoints
  • Cell Line
  • Cytoprotection
  • Gene Expression Regulation
  • Gene Knockout Techniques
  • Humans
  • Hyaluronan Receptors
  • Hyaluronic Acid
  • Longevity
  • Mice
  • Mole Rats
  • Molecular Weight
  • Primary Cell Culture
  • Protein Interaction Maps
  • RNA-Seq
  • Signal Transduction
  • Species Specificity
  • Stress, Physiological
  • Tumor Suppressor Protein p53


Maturity-dependent cartilage cell plasticity and sensitivity to external perturbation.

Articular cartilage undergoes biological and morphological changes throughout maturation. The prevalence of osteoarthritis in the aged population suggests that maturation predisposes cartilage to degradation and/or impaired regeneration, but this process is not fully understood. Therefore, the objective of this study was to characterize the cellular and genetic profile of cartilage, as well as biological plasticity in response to mechanical and culture time stimuli, as a function of animal maturity. Porcine articular cartilage explants were harvested from stifle joints of immature (2-4 weeks), adolescent (5-6 months), and mature (1-5 years) animals. Half of all samples were subjected to a single compressive mechanical load. Loaded samples were paired with unloaded controls for downstream analyses. Expression of cartilage progenitor cell markers CD105, CD44, and CD29 were determined via flow cytometry. Expression of matrix synthesis genes Col1, Col2, Col10, ACAN, and SOX9 were determined via qPCR. Tissue morphology and matrix content were examined histologically. Post-loading assays were performed immediately and following 7 days in culture. CD105 and CD29 expression decreased with maturity, while CD44 expression was upregulated in cartilage from mature animals. Expression of matrix synthesis genes were generally upregulated in cartilage from mature animals, and adolescent animals showed the lowest expression of several matrix synthesizing genes. Culture time and mechanical loading analyses revealed greater plasticity to mechanical loading and culture time in cartilage from younger animals. Histology confirmed distinct structural and biochemical profiles across maturity. This study demonstrates differential, nonlinear expression of chondroprogenitor markers and matrix synthesis genes as a function of cartilage maturity, as well as loss of biological plasticity in aged tissue. These findings have likely implications for age-related loss of regeneration and osteoarthritis progression.


Keywords

  • Aging
  • Articular cartilage
  • Osteoarthritis
  • Plasticity
  • Progenitor cells


Aged Mice Exhibit Severe Exacerbations of Dry Eye Disease with an Amplified Memory Th17 Cell Response.

The prevalence as well as the severity of dry eye disease increase with age. Memory T helper 17 (Th17) cells (CD4 IL-17A CD44 ) drive the chronic and relapsing course of dry eye disease. Here, we investigated the contribution of memory Th17 cells to age-related dry eye disease, and evaluated memory Th17 cell depletion with anti-IL-15 antibody as a strategy to abrogate the severe exacerbations of dry eye disease observed in aged mice. After initial exposure to desiccating stress, aged mice maintained higher frequencies of memory Th17 cells in the draining lymph nodes relative to young mice. Upon secondary exposure to desiccating stress, aged mice developed more severe corneal epitheliopathy than young mice, which is associated with increased local frequencies of Th17 cells (CD4 IL-17A ). Treatment with anti-IL-15 antibody decreased the enlarged memory Th17 pool in aged mice to frequencies comparable with young mice. Furthermore, anti-IL-15-treated mice showed significantly reduced conjunctival infiltration of Th17 cells and lower corneal fluorescein staining scores compared with saline-treated control mice. Our data suggest that age-related increases in the memory Th17 compartment predispose aged mice toward the development of severe corneal epithelial disease after exposure to a dry environment. Selectively targeting memory Th17 cells may be a viable therapeutic approach in the treatment of age-related dry eye disease.

MeSH Terms

  • Aging
  • Animals
  • Dry Eye Syndromes
  • Female
  • Immunologic Memory
  • Mice
  • Mice, Inbred C57BL
  • Th17 Cells


Effects of continuous exposure to low concentration of ClO gas on the growth, viability, and maintenance of undifferentiated MSCs in long-term cultures.

Hygienic management is more important in the manufacturing of cell products than in the production of chemical agents, because cell material and final product cannot be decontaminated. On the other hand, especially in the selection of hygienic agent, the adverse effects on the cells must be considered as well as the decontamination effect. ClO is a potent disinfectant, which is now expected as a safe and effective hygienic agent in the field of cell production. In this study, we investigated the effects of low dose ClO gas in the atmosphere of CO incubator on the characteristics of MSCs cultured in it. First, we installed a ClO generator to a CO incubator for cell culture in which a constant level of ClO can be maintained. After culturing human cord derived MSCs in the CO incubator, the characteristics of cells were analyzed. Continuous exposure to 0.05 ppmv of ClO gas did not affect cell proliferation until at least 8th passage. In the FACS analysis, antigens usually expressed on MSCs, CD105, CD90, CD44, CD73 and CD29, were positively observed, but differentiation markers, CD11b and CD34, were little expressed on the MSCs exposed to 0.05 ppmv or 0.1 ppmv of ClO gas just as on the control cells. Also in the investigation for cell death, 0.05 ppmv and 0.1 ppmv of ClO gas little affected the viability, apoptosis or necrosis of MSCs. Furthermore, we assessed senescence using SA-β-gal staining. Although the frequency of stained cells cultured in 0.1 ppmv of ClO gas was significantly increased than that of not exposed cells, the stained cells in 0.05 ppmv were rare and their frequency was almost the same as that in control. All these results indicate that, although excessive concentration of ClO gas induces senescence but neither apoptosis nor cell differentiation, exposure to 0.05 ppmv of ClO gas little affected the characteristics of MSCs. In this study we demonstrate that continuous exposure to appropriate dose of ClO gas can be safely used as decontamination agent in cell processing facilities.


Keywords

  • Cell processing
  • Chlorine dioxide (ClO2)
  • ClO2, chlorine dioxide
  • EPA, Environmental Protection Agency
  • FDA, Food and Drug Administration
  • H2O2, hydrogen peroxide
  • HEPA, high efficiency particulate air
  • Hygienic management
  • MSCs, mesenchymal stem cells
  • Mesenchymal stem cells (MSCs)
  • OSHA, Occupational Safety and Health Administration
  • PMD Act, Pharmaceuticals and Medical Devices Act
  • Senescence
  • TWA, time weight average
  • WHO, World Health Organization


Chronic circadian misalignment accelerates immune senescence and abbreviates lifespan in mice.

Modern society characterized by a 24/7 lifestyle leads to misalignment between environmental cycles and endogenous circadian rhythms. Persisting circadian misalignment leads to deleterious effects on health and healthspan. However, the underlying mechanism remains not fully understood. Here, we subjected adult, wild-type mice to distinct chronic jet-lag paradigms, which showed that long-term circadian misalignment induced significant early mortality. Non-biased RNA sequencing analysis using liver and kidney showed marked activation of gene regulatory pathways associated with the immune system and immune disease in both organs. In accordance, we observed enhanced steatohepatitis with infiltration of inflammatory cells. The investigation of senescence-associated immune cell subsets from the spleens and mesenteric lymph nodes revealed an increase in PD-1 [[CD44]] CD4 T cells as well as CD95 GL7 germinal center B cells, indicating that the long-term circadian misalignment exacerbates immune senescence and consequent chronic inflammation. Our results underscore immune homeostasis as a pivotal interventional target against clock-related disorders.

MeSH Terms

  • Animals
  • B-Lymphocytes
  • Cellular Senescence
  • Circadian Rhythm
  • Disease Models, Animal
  • Humans
  • Hyaluronan Receptors
  • Inflammation
  • Jet Lag Syndrome
  • Longevity
  • Mice
  • Programmed Cell Death 1 Receptor
  • Sequence Analysis, RNA
  • T-Lymphocytes


Characterization of human telomerase reverse transcriptase immortalized anterior cruciate ligament cell lines.

The anterior-cruciate-ligament (ACL) contains mesenchymal stem cells (ACL-MSCs), suggesting the feasibility of regenerative treatments of this tissue. The immortalization of isolated cells results in cell-lines applicable to develop cell-based therapies. Immortal cell lines eliminate the need for frequent cell isolation from donor tissues. The objective of this study was to characterize cell lines that were generated from isolated ACL-MSCs using TERT gene transfer. We isolated ACL-MSCs from human ACLs derived at the time of ACL reconstruction surgery or total knee arthroplasty. We generated cell lines and compared them to non-immortalized ACL-MSCs. We assessed the cellular morphology and we detected surface antigen expression. The resistance to senescence was inferred using the beta galactosidase activity. Histology, immunohistochemistry, and reverse transcriptase polymerase chain reaction (RT-PCR) were used to evaluate the multilineage differentiation capacity. The morphology of hTERT-ACL-MSCs was similar to ACL up to the last assessment at passage eight. We detected a strong surface expression of CD44, CD90, CD105, and STRO-1 in hTERT-ACL-MSCs. No substantial reduction in the ATP activity was observed in hTERT-ACL-MSCs. Cell lines generated from ACL-MSCs maintain their morphology, surface antigen expression profile, and proliferative capacity; while markers of senescence appear to be reduced. These cell-lines maintained their multilineage differentiation capacity. The demonstrated model systems can be used for further development of new cell-based regenerative approaches in anterior cruciate ligament research, which may lead to new therapeutic strategies in the future.

MeSH Terms

  • Adolescent
  • Aged
  • Anterior Cruciate Ligament
  • Cell Differentiation
  • Cell Separation
  • Cells, Cultured
  • Humans
  • Mesenchymal Stem Cells
  • Telomerase

Keywords

  • Anterior cruciate ligament
  • Immortalization
  • Mesenchymal stem cells
  • Multilineage differentiation
  • Senescence


Effect of Allogenic Bone Marrow Mesenchymal Stem Cell Transplantation on T Cells of Old Mice.

To evaluate age-related changes in T cells and stem cell-related genes in mice and the changes in T cells and stem cell-related genes after allogenic bone marrow mesenchymal stem cell (BMSC) transplantation, BALB/c mice were divided into young (2 months, [i]n[/i] = 5) and old (20 months, [i]n[/i] = 5) groups and 1 × 10 BMSCs from 3-week-old C57BL/6J mice were injected into the old mice ([i]n[/i] = 5). T lymphocytes including CD3 , CD8 , CD8 CD28 , and CD8 CD44 CD62L Sca-1 stem cell-like memory T cells from spleens were analyzed by flow cytometry. mRNA transcriptions of the tumor suppressor [i]p16 [/i] and the senescence inhibiting [i]AUF 1[/i] and stem cell-related [i]ADAM12[/i], [i]GIL3[/i], [i]c-MYC[/i], [i]NANOG[/i], [i]Wnt[/i], [i]HOX11[/i], [i]Sox2[/i], [i]Oct3/4[/i], and [i]KLF4[/i] genes were analyzed by quantitative reverse transcription-polymerase chain reaction for comparison between young and old mice and old mice after BMSC application. Stem cell-related genes were reduced transcribed in old mice, an action that could be partly or completely reversed for some genes by BMSC injections. The proportion of CD8 CD28 T cells in the spleens of old mice was significantly reduced ([i]p[/i] < 0.01), indicating advanced proliferative T cell senescence. The CD8 CD44 CD62L cell fraction was significantly reduced and that of CD8 CD44 CD62L Sca-1 increased in splenic CD8 cells of old mice, both actions of which were reversed by BMSC injections. [i]p16 [/i] transcription was enhanced and [i]AUF1[/i] transcription was reduced in old mice, the latter effect partly reversed by BMSC injections. BMSC injections led to recovery of stem cell-related gene activation or BMSC stem cell-related gene expression tolerance in spleens of old mice.


Keywords

  • aging
  • cellular senescence
  • memory T cells
  • stem cell


The Hyaluronidase, TMEM2, Promotes ER Homeostasis and Longevity Independent of the UPR .

Cells have evolved complex mechanisms to maintain protein homeostasis, such as the UPR , which are strongly associated with several diseases and the aging process. We performed a whole-genome CRISPR-based knockout (KO) screen to identify genes important for cells to survive ER-based protein misfolding stress. We identified the cell-surface hyaluronidase (HAase), Transmembrane Protein 2 (TMEM2), as a potent modulator of ER stress resistance. The breakdown of the glycosaminoglycan, hyaluronan (HA), by TMEM2 within the extracellular matrix (ECM) altered ER stress resistance independent of canonical UPR pathways but dependent upon the cell-surface receptor, CD44, a putative HA receptor, and the MAPK cell-signaling components, ERK and p38. Last, and most surprisingly, ectopic expression of human TMEM2 in C. elegans protected animals from ER stress and increased both longevity and pathogen resistance independent of canonical UPR activation but dependent on the ERK ortholog mpk-1 and the p38 ortholog pmk-1.

MeSH Terms

  • Animals
  • Caenorhabditis elegans
  • Cell Line
  • Cell Proliferation
  • Disease Resistance
  • Endoplasmic Reticulum
  • Endoplasmic Reticulum Stress
  • Fibroblasts
  • Humans
  • Hyaluronoglucosaminidase
  • Immunity, Innate
  • Longevity
  • Membrane Proteins
  • Models, Biological
  • Molecular Weight
  • Signal Transduction
  • Unfolded Protein Response

Keywords

  • CRISPR-Cas9
  • MAPK signaling
  • aging
  • endoplasmic reticulum
  • extracellular matrix
  • glucosaminoglycan
  • immune response
  • stress response


Defective induction of the proteasome associated with T-cell receptor signaling underlies T-cell senescence.

The proteasome degradation machinery is essential for a variety of cellular processes including senescence and T-cell immunity. Decreased proteasome activity is associated with the aging process; however, the regulation of the proteasome in CD4 T cells in relation to aging is unclear. Here, we show that defects in the induction of the proteasome in CD4 T cells upon T-cell receptor (TCR) stimulation underlie T-cell senescence. Proteasome dysfunction promotes senescence-associated phenotypes, including defective proliferation, cytokine production and increased levels of PD-1 [[CD44]] CD4 T cells. Proteasome induction by TCR signaling via MEK-, IKK- and calcineurin-dependent pathways is attenuated with age and decreased in PD-1 [[CD44]] CD4 T cells, the proportion of which increases with age. Our results indicate that defective induction of the proteasome is a hallmark of CD4 T-cell senescence.

MeSH Terms

  • Animals
  • CD4-Positive T-Lymphocytes
  • Cell Proliferation
  • Cells, Cultured
  • Cellular Senescence
  • Cytokines
  • Hyaluronan Receptors
  • Mice
  • Mice, Inbred C57BL
  • Phenotype
  • Programmed Cell Death 1 Receptor
  • Proteasome Endopeptidase Complex
  • Receptors, Antigen, T-Cell
  • Signal Transduction

Keywords

  • T cell receptor signal
  • T cell senescence
  • aging
  • proteasome


CD44v8-10 and CD44s Are Age-dependently Expressed in Primary Cultured Papillary Thyroid Carcinoma Cells and Are Associated with Cell Proliferation.

Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, arising from follicular cells, and accounts for more than 80% of all thyroid malignant tumors. Although age is the strongest prognostic factor of PTC, and various cut-off ages (40-55 years) were suggested in previous studies, the molecular mechanisms causing age-related changes of PTC cell proliferation remain unclear. CD44 is a major cell surface receptor for hyaluronate and is known as a cancer stem cell marker. However, the association between CD44 and PTC is still unknown. Therefore, we determined the proliferation of primary cultured cells obtained from patients with PTC, and the CD44 mRNA expression profile to elucidate age-related association of CD44 with PTC. The results showed that cell proliferation was significantly decreased according to age. We also found that CD44v8-10 and CD44 splice variants were expressed dominantly in patients with PTC. Moreover, the CD44v8-10/CD44s mRNA expression ratio was significantly increased according to age, and there was a significant negative correlation between this expression ratio and cell proliferation. Our findings suggest that the CD44v8-10/CD44s expression ratio in PTC cells is useful for screening for aggressive PTC and may provide clinically valuable information.

MeSH Terms

  • Adolescent
  • Adult
  • Age Factors
  • Aged
  • Cell Line, Tumor
  • Cell Proliferation
  • Female
  • Humans
  • Hyaluronan Receptors
  • Male
  • Middle Aged
  • RNA, Messenger
  • Thyroid Cancer, Papillary
  • Young Adult

Keywords

  • Aging
  • CD44
  • Papillary thyroid carcinoma
  • Splice variant


Immunophenotypic characterization, multi-lineage differentiation and aging of zebrafish heart and liver tissue-derived mesenchymal stem cells as a novel approach in stem cell-based therapy.

Mesenchymal stem cells (MSCs) are a good model for preclinical and clinical investigations, and alternative sources of MSCs are subject to intensive experiments. In this study, mesenchymal stem cells (MSCs) were isolated from heart and liver tissue of Zebrafish (Danio rerio). The flow-cytometry as well as RT-PCR were used to analyze the expression of a panel of cell surface markers CD44, CD90, CD31 and CD34. In the following, alizarin red, oil red-O and toluidine blue staining were carried out to evaluate the multi-lineage differentiation of zebrafish heart and liver tissue-derived MSCs. Subsequently, the gene and protein expression of Oct4, Sox2 and Nanog as pluri-potent markers were analyzed by RT-PCR and western blotting, respectively. In addition, MTT assay was used for cell proliferation potential and population doubling time (PDT) assessment. Also, the aging of cells was investigated by β-galactosidase activity assay. The results showed that, like other MSCs, zebrafish heart and liver tissue-derived MSCs were positive for mesenchymal, negative for hematopoietic markers and expressed pluripotent markers Oct4, Sox2 and Nanog. Moreover, these cells were differentiated to osteocyte, adipocyte, and chondrocyte lineages following directed differentiation. It was found that PDT of zebrafish heart and liver tissue-derived MSCs were 50.67 and 46.61 h, respectively. These cells had significantly more rapid growth on day 4. Our results show that zebrafish heart and liver tissue-derived MSCs exhibited typical MSC characteristics including fibroblast morphology, multi-lineage differentiation capacity, pluripotency potential and expression of a typical set of classic MSC surface markers.

MeSH Terms

  • Aging
  • Animals
  • Cell Differentiation
  • Cell Lineage
  • Cell Proliferation
  • Heart
  • Immunophenotyping
  • Liver
  • Mesenchymal Stem Cells
  • Myocardium
  • Regenerative Medicine
  • Zebrafish

Keywords

  • Characterization
  • Multi-lineage differentiation
  • Regenerative medicine
  • Zebrafish heart and liver tissue-derived MSCs


CD8 , but not CD4 effector/memory T cells, express the [[CD44]] CD45RB phenotype with aging, which displays reduced expression levels of P2X receptor and ATP-induced cellular responses.

Previously we reported that the sensitivity of CD4 T cells to ATP does not depend on P2X receptor (P2X R) expression levels but on their activation and differentiation stages. Therefore, here we have investigated a potential relationship between the sensitivity of CD8 T cells to ATP and their stages of differentiation. Thus, the CD8 subpopulation exhibits a drastically reduced sensitivity to ATP with aging, which parallels the strong increase of an effector/memory CD8 subset expressing high levels of [[CD44]] cell adhesion molecule and CD45RB transmembrane phosphatase ([[CD44]] CD45RB ). Using l-selectin/CD62L, CC-chemokine receptor 7, and CD127/IL-7 receptor-α markers, we showed that effector/memory CD8 T cells belong to a central or effector memory subset. In contrast, the [[CD44]] CD45RB effector/memory subset is absent or poorly expressed in the CD4 T subpopulation regardless of age. While ATP treatment can trigger channel and pore formation, CD62L shedding, phosphatidylserine exposure, and cell death in the [[CD44]] CD45RB -naive CD8 subset, it is unable to induce these cellular activities in the [[CD44]] CD45RB effector/memory CD8 subset. Importantly, both [[CD44]] CD45RB -naive and [[CD44]] CD45RB effector/memory subsets express similar low levels of P2X R, demonstrating that the sensitivity of CD8 T cells to ATP depends on the stage of differentiation instead of P2X R expression levels.-Mellouk, A., Bobé, P. CD8 , but not CD4 effector/memory T cells, express the [[CD44]] CD45RB phenotype with aging, which displays reduced expression levels of P2X receptor and ATP-induced cellular responses.

MeSH Terms

  • Adenosine Triphosphate
  • Aging
  • Animals
  • CD4-Positive T-Lymphocytes
  • CD8-Positive T-Lymphocytes
  • Calcium Signaling
  • Cell Differentiation
  • Hyaluronan Receptors
  • Immunologic Memory
  • L-Selectin
  • Leukocyte Common Antigens
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Phenotype
  • Receptors, Purinergic P2X7
  • T-Lymphocyte Subsets

Keywords

  • T lymphocyte
  • naive T lymphocyte
  • purinergic signaling


Regenerative potential of human nucleus pulposus resident stem/progenitor cells declines with ageing and intervertebral disc degeneration.

Numerous studies have demonstrated the presence of resident nucleus pulposus stem/progenitor cells (NPSCs) in the tissue of the intervertebral disc (IVD). However, the cellular identity of NPSCs during IVD degeneration and ageing are poorly defined at present, despite significant progress in the understanding of NPSC biology. In the present study, NPSCs were isolated from human degenerated IVD and were characterized by flow cytometry, gene expression assays and proliferation and multipotency analysis. The results of the present study demonstrated that NPSCs isolated from human degenerated IVD may be divided into two groups according to the expression of mesenchymal stem cell (MSC) surface markers: The high expression of MSC surface markers group (H‑NPSCs) was highly positive for CD29, CD44, CD73, CD90 and CD105 at rates >95%, and the low expression of MSC markers surface markers group (L‑NPSCs), with the expression of CD29 and CD105 exhibiting individual variability, however, all at rates <95%. The donors for H‑NPSCs were aged <20 years, while the majority of donors for L‑NPSCs were aged >25 years, with one exception aged <20 years. The results highlighted that the low expression of MSC surface markers in NPSCs from aged and degenerated NP tissues were associated with a low rate of proliferation and reduced differentiation potential, as well as downregulation of the NP progenitor marker Tie2 and higher expression of NP cell‑specific markers. These findings demonstrated that the regenerative potential of human NPSCs declines with ageing and degeneration of the IVD.

MeSH Terms

  • Adolescent
  • Adult
  • Aging
  • Antigens, CD
  • Cell Differentiation
  • Female
  • Humans
  • Intervertebral Disc
  • Intervertebral Disc Degeneration
  • Male
  • Regeneration
  • Stem Cells


Multi-color flow cytometry for evaluating age-related changes in memory lymphocyte subsets in dogs.

While dogs are increasingly being utilized as large-animal models of disease, important features of age-related immunosenescence in the dog have yet to be evaluated due to the lack of defined naïve vs. memory T lymphocyte phenotypes. We therefore performed multi-color flow cytometry on peripheral blood mononuclear cells from young and aged beagles, and determined the differential cytokine production by proposed memory subsets. CD4 and CD8 T lymphocytes in aged dogs displayed increased cytokine production, and decreased proliferative capacity. Antibodies targeting CD45RA and CD62L, but less so CD28 or CD44, defined canine cells that consistently exhibited properties of naïve-, central memory-, effector memory-, and terminal effector-like CD4 and CD8 T lymphocyte subsets. Older dogs demonstrated decreased frequencies of naïve-like CD4 and CD8 T lymphocytes, and an increased frequency of terminal effector-like CD8 T lymphocytes. Overall findings revealed that aged dogs displayed features of immunosenescence similar to those reported in other species.

MeSH Terms

  • Aging
  • Animals
  • CD4-Positive T-Lymphocytes
  • CD8-Positive T-Lymphocytes
  • Cells, Cultured
  • Cytokines
  • Dogs
  • Flow Cytometry
  • Immunologic Memory
  • L-Selectin
  • Leukocyte Common Antigens
  • Leukocytes, Mononuclear
  • Lymphocyte Subsets
  • Male

Keywords

  • CD45RA
  • Canine
  • Immunosenescence
  • Inflammaging
  • T lymphocyte


Therapeutic efficacy of olfactory stem cells in rotenone induced Parkinsonism in adult male albino rats.

Olfactory stem cells (OSCs) are found in the olfactory mucosa and olfactory bulb and have the capacity to proliferate and differentiate along multiple tissue lineages. Rotenone; widely used insecticide has a neurodegenerative effect on the dopaminergic cells of substantia nigra (SN) of midbrain producing Parkinsonism. The aim of this study is to isolate rat OSCs from olfactory mucosa and olfactory bulb, culture these OSCs in suitable medium to allow for their proliferation to be used in the treatment of Parkinsonism induced by rotenone. The characteristics of OSCs, the effects of rotenone on the SN of midbrain and the curative effect of OSCs on the substantia nigra were determined morphologically, immunohistochemically, and by transmission electron microscopy. PKH 26; immunofluorescent dye was used as a cell tracer to locate the transplanted cells in host midbrain. OSCs were spindle shaped with irregular processes, and were positive for CD44 and Nestin and negative for CD34. Subcutaneous rotenone produced Parkinsonism through producing degeneration of the dopaminergic cells of SN of the midbrain. Transplantation of OSCs produced restoration of the normal structure of SN and dopaminergic cells and improves the clinical manifestations of Parkinsonism. These results indicate that, the isolated rat OSCs can proliferate and expand in vitro when culture in suitable medium and these cells can exert therapeutic effects in Parkinsonism by recruitment in SN and restoration of the structure and function of dopaminergic cells.

MeSH Terms

  • Aging
  • Animals
  • Cells, Cultured
  • Male
  • Mesencephalon
  • Motor Activity
  • Olfactory Bulb
  • Parkinsonian Disorders
  • Rats
  • Rotenone
  • Stem Cell Transplantation
  • Stem Cells
  • Treatment Outcome
  • Tyrosine 3-Monooxygenase

Keywords

  • Characterization
  • Isolation
  • Olfactory stem cells
  • Parkinsonism
  • Rotenone


Precise Intradermal Injection of Nanofat-Derived Stromal Cells Combined with Platelet-Rich Fibrin Improves the Efficacy of Facial Skin Rejuvenation.

The rejuvenation properties of nanofat grafting have been described in recent years. However, it is not clear whether the clinical efficacy of the procedure is attributable to stem cells or linked to other components of adipose tissue. In this study we isolated nanofat-derived stem cells (NFSCs) to observe their biological characteristics and evaluate the efficacy of precise intradermal injection of nanofat combined with platelet-rich fibrin (PRF) in patients undergoing facial rejuvenation treatment. Third-passage NFSCs were isolated and cultured using a mechanical emulsification method and their surface CD markers were analyzed by flow cytometry. The adipogenic and osteogenic nature and chondrogenic differentiation capacity of NFSCs were determined using Oil Red O staining, alizarin red staining, and Alcian blue staining, respectively. Paracrine function of NFSCs was evaluated by enzyme-linked immunosorbent assay (ELISA) at 1, 3, 7, 14, and 28 days after establishing the culture. Then, the effects of PRF on NFSC proliferation were assessed in vitro. Finally, we compared the outcome in 103 patients with facial skin aging who underwent both nanofat and intradermal PRF injection (treatment group) and 128 patients who underwent hyaluronic acid (HA) injection treatment (control group). Outcomes in the two groups were compared by assessing pictures taken at the same angle before and after treatment, postoperative recovery, incidence of local absorption and cysts, and skin quality before treatment, and at 1, 12, 24 months after treatment using the VISIA Skin Image Analyzer and a SOFT5.5 skin test instrument. NFSCs expressed CD29, CD44, CD49d, CD73, CD90, and CD105, but did not express CD34, CD45, and CD106. NFSCs also differentiated into adipocytes, osteoblasts, and chondrocytes under appropriate induction conditions. NFSCs released large amounts of growth factors such as VEGF, bFGF, EGF, and others, and growth factor levels increased in a time-dependent manner. At the same time, PRF enhanced proliferation of NFSCs in vitro in a dose-dependent manner, and the growth curves under different concentrations of PRF all showed plateaus 6d after seeding. Facial skin texture was improved to a greater extent after combined injection of nanofat and PRF than after control injection of HA. The nanofat-PRF group had a higher satisfaction rate. Neither treatment caused any complications such as infection, anaphylaxis, or paresthesia during long-term follow-up. NFSCs demonstrate excellent multipotential differentiation and paracrine function, and PRF promotes proliferation of NFSCs during the early stage after seeding. Both nanofat-PRF and HA injection improve facial skin status without serious complications, but the former was associated with greater patient satisfaction, implying that nanofat-PRF injection is a safe, highly effective, and long-lasting method for skin rejuvenation.

MeSH Terms

  • Adipose Tissue
  • Adult
  • Cell Proliferation
  • Cells, Cultured
  • Face
  • Female
  • Humans
  • Injections, Intradermal
  • Intercellular Signaling Peptides and Proteins
  • Male
  • Middle Aged
  • Platelet-Rich Fibrin
  • Rejuvenation
  • Skin Aging
  • Skin Physiological Phenomena
  • Stromal Cells
  • Young Adult

Keywords

  • Facial skin aging
  • Nanofat-derived stem cells
  • Platelet-rich fibrin
  • Rejuvenation
  • Stromal vascular fraction


Some chemotherapeutics-treated colon cancer cells display a specific phenotype being a combination of stem-like and senescent cell features.

Colorectal cancer (CRC) is the second leading cause of death among cancer patients in the Northern countries. CRC can reappear a long time after treatment. Recent clinical studies demonstrated that, in response to chemotherapy, cancer cells may undergo stress-induced premature senescence (SIPS), which typically results in growth arrest. Nonetheless, these senescent cells were reported to divide in an atypical manner and thus contribute to cancer re-growth. Therefore, we examined if SIPS escape may follow treatment with chemotherapeutics used clinically: 5-fluorouracil (5-FU), oxaliplatin (OXA) and irinotecan (IRINO). To mimic the therapeutic regimes we exposed human colon cancer HCT116 and SW480 cells to repeated cycles of drug treatment. The cells treated with 5-FU or IRINO exhibited several hallmarks of SIPS: growth arrest, increased size and granularity, polyploidization, augmented activity of the SA-β-galactosidase, accumulation of P21 and CYCLIN D1 proteins, and the senescence-associated secretory phenotype. Moreover, re-population of the cancer cell cultures was delayed upon treatment with the senescence-inducing agents. At the same time, we detected a subpopulation of senescent colon cancer cells with features of stemness: elevated NANOG expression, exclusion of Hoechst 33342 (typical for side population) and increased CD24 expression. Additionally, rare, polyploid cells exhibited blastocyst-like morphology and produced progeny. In parallel, majority of chemotherapeutics-treated cells underwent mesenchymal to epithelial transition, as the percentage of CD44-positve cells was reduced, and levels of E-cadherin (epithelial marker) were elevated. Our study demonstrates that a subpopulation of chemotherapeutics-treated colon cancer cells display a specific phenotype being a combination of stem-like and senescent cell features. This may contribute to their resistance to chemotherapy and their ability to re-grow cancer after completion of therapeutic intervention.

MeSH Terms

  • Antineoplastic Agents
  • Cellular Senescence
  • Colonic Neoplasms
  • Cyclin D1
  • Drug Resistance, Neoplasm
  • Epithelial-Mesenchymal Transition
  • Fluorouracil
  • HCT116 Cells
  • Humans
  • Hyaluronan Receptors
  • Irinotecan
  • Neoplastic Stem Cells
  • Oxaliplatin

Keywords

  • 5-fluoruracil
  • angiogenesis
  • cancer Biology
  • cancer stem cells
  • cell Cycle control
  • chemotherapy
  • colon cancer
  • doxorubicin
  • irinotecan
  • molecular Therapy
  • oxaliplatin
  • senescence


A Trickster in Disguise: Hyaluronan's Ambivalent Roles in the Matrix.

Hyaluronan (HA) is a simple but diverse glycosaminoglycan. It plays a major role in aging, cellular senescence, cancer, and tissue homeostasis. In which way HA affects the surrounding tissues greatly depends on the molecular weight of HA. Whereas high molecular weight HA is associated with homeostasis and protective effects, HA fragments tend to be linked to the pathologic state. Furthermore, the interaction of HA with its binding partners, the hyaladherins, such as CD44, is essential for sustaining tissue integrity and is likewise related to cancer. The naked mole rat, a rodent species, possesses a special form of very high molecular weight (vHMW) HA, which is associated with the extraordinary cancer resistance and longevity of those animals. This review addresses HA and its diverse facets: from HA synthesis to degradation, from oligomeric HA to vHMW-HA and from its beneficial properties to the involvement in pathologies. We further discuss the functions of HA in the naked mole rat and compare them to human conditions. Though intensively researched, this simple polymer bears some secrets that may hold the key for a better understanding of cellular processes and the development of diseases, such as cancer.


Keywords

  • CD44
  • aging
  • cancer
  • cancer resistance
  • cellular senescence
  • early contact inhibition
  • hyaluronan
  • naked mole rat


Longevity effect of a polysaccharide from Chlorophytum borivilianum on Caenorhabditis elegans and Saccharomyces cerevisiae.

The traditional Indian medicine, Ayurveda, provides insights and practical solutions towards a healthy life style. Rasayana is a branch of Ayurveda known for preserving and promoting health, enhancing the quality of life and delaying the aging process. In the traditional knowledge, the Rasayana herb, Chlorophytum borivilianum (C. borivilanum) is regarded as a general health promoting tonic that delays aging and increases lifespan, cognitive function and physical strength. Aging is a complex and multifactorial physiological phenomenon that manifests itself over a wide range of biological systems, tissues, and functions. Longevity is an obvious marker of physiological aging. Simple model systems such as the single-cell budding yeast Saccharomyces cerevisiae (S. cerevisiae) and the nematode, Caenorhabditis elegans (C. elegans) are widely used to study the aging process and longevity. Here, we show that a polysaccharide fraction obtained from C. borivilianum increases the lifespan of S. cerevisiae and C. elegans, using an automated screening platform (ChronoscreenTM). Chemical analysis of this extract revealed a low molecular weight polysaccharide of 1000 Da, predominantly comprising Glu1→6Glu linkage. This polysaccharide showed significant dose-dependent extension of the median lifespan of S. cerevisiae by up to 41% and of the median lifespan of C. elegans by up to 10%. Taking cue from these results and the traditionally described benefits of Rasayanas on skin rejuvenation, we tested in vitro the polysaccharide for potential skin benefits. In a keratinocyte culture, we observed that this polysaccharide increased cell proliferation significantly, and induced synthesis of hyaluronic acid (HA), a well-known extracellular matrix component. Furthermore, when added to culture medium of human reconstructed epidermis, we observed an enhanced production of epidermal markers, e.g. CD44 and HA that are otherwise diminished in aged skin. Together, these results suggest that in addition to life-span extension of S. cerevisiae and C. elegans, a polysaccharide from the Rasayana herb, C. borivilianum may have beneficial effects on skin aging parameters.

MeSH Terms

  • Aging
  • Animals
  • Asparagaceae
  • Caenorhabditis elegans
  • Cell Proliferation
  • Dose-Response Relationship, Drug
  • Keratinocytes
  • Longevity
  • Medicine, Ayurvedic
  • Plant Extracts
  • Polysaccharides
  • Saccharomyces cerevisiae


Physiologic Thymic Involution Underlies Age-Dependent Accumulation of Senescence-Associated CD4 T Cells.

Immune aging may underlie various aging-related disorders, including diminished resistance to infection, chronic inflammatory disorders, and autoimmunity. PD-1 and CD153 [[CD44]] CD4 T cells with features of cellular senescence, termed senescence-associated T (SA-T) cells, increasingly accumulate with age and may play a role in the immune aging phenotype. In this article, we demonstrate that, compared with young mice, the aged mouse environment is highly permissive for spontaneous proliferation of transferred naive CD4 T cells, and it drives their transition to PD-1 and CD153 [[CD44]] CD4 T cells after extensive cell divisions. CD4 T cells with essentially the same features as SA-T cells in aged mice are also generated from naive CD4 T cells after extensive cell divisions under severe T-lymphopenic conditions by gamma irradiation or in developmental T cell defect, often in association with spontaneous germinal centers, as seen in aged mice. The increase in SA-T cells is significantly enhanced after thymectomy at the young adult stage, along with accelerated T cell homeostatic proliferation, whereas embryonic thymus implantation in the late adult stage markedly restricts the homeostatic proliferation of naive CD4 T cells in the host and delays the increase in SA-T cells. Our results suggest that reduced T cell output due to physiologic thymic involution underlies the age-dependent accumulation of SA-T cells as a result of increasing homeostatic proliferation of naive CD4 T cells. SA-T cells may provide a suitable biomarker of immune aging, as well as a potential target for controlling aging-related disorders.

MeSH Terms

  • Aging
  • Animals
  • Autoimmunity
  • Biomarkers
  • CD30 Ligand
  • CD4-Positive T-Lymphocytes
  • Cell Differentiation
  • Cellular Senescence
  • Germinal Center
  • Hyaluronan Receptors
  • Lymphocyte Activation
  • Mice
  • Phenotype
  • Programmed Cell Death 1 Receptor
  • Thymus Gland


CD44 alveolar type II cells show stem cell properties during steady-state alveolar homeostasis.

The alveolar epithelium is composed of type I cells covering most of the gas-blood exchange surface and type II cells secreting surfactant that lowers surface tension of alveoli to prevent alveolar collapse. Here, we have identified a subgroup of type II cells expressing a higher level of cell surface molecule CD44 (CD44 type II cells) that composed ~3% of total type II cells in 5-10-wk-old mice. These cells were preferentially apposed to lung capillaries. They displayed a higher proliferation rate and augmented differentiation capacity into type I cells and the ability to form alveolar organoids compared with CD44 type II cells. Moreover, in aged mice, 18-24 mo old, the percentage of CD44 type II cells among all type II cells was increased, but these cells showed decreased progenitor properties. Thus CD44 type II cells likely represent a type II cell subpopulation important for constitutive regulation of alveolar homeostasis.

MeSH Terms

  • Aging
  • Alveolar Epithelial Cells
  • Animals
  • Capillaries
  • Cell Differentiation
  • Cell Lineage
  • Cell Proliferation
  • Cells, Cultured
  • Homeostasis
  • Hyaluronan Receptors
  • Integrins
  • Lung
  • Mice, Inbred C57BL
  • Organoids
  • Pulmonary Surfactant-Associated Protein C
  • Stem Cells

Keywords

  • CD44
  • alveoli
  • homeostasis
  • lung
  • type II cells


Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line.

There is compelling evidence associating senescent cells with the malignant progression of tumours. Of all senescence-related mechanisms, the so-called senescence-associated secretory phenotype (SASP) has attracted much attention. Since the pro-inflammatory cytokines IL-6 and IL-8 are consistently present in the SASP, and secreted by highly aggressive breast cancer cell lines, we aimed at elucidating their role on the less aggressive breast cancer cell line MCF-7, which does not secret these cytokines. The MCF-7 cell line was treated with either senescence-conditioned medium (SCM), IL-6 or IL-8 and then evaluated for phenotypic (CD44 and CD24 by FACS) and functional changes associated with an EMT program (migration/invasion) and for the acquisition of stem cell properties: mammosphere-forming capacity, expression of reprogramming factors (by qRT-PCR) and multilineage differentiation potential. We also evaluated the role of IL6 and IL8 in the cytokine-secreting, highly tumorigenic cell line MDA-MB-231. Our results show that treatment of MCF-7 cells with IL6 and IL8, alone or together, induced the appearance of cells with fibroblastoid morphology, increased CD44 expression and migration, self-renewal and multilineage differentiation capacity, all characteristics compatible with an EMT program and stemness. These changes closely resembled those induced by a SCM. Interestingly, SCM treatments further increased IL6 and IL8 secretion by MCF-7 cells, thus suggesting the participation of an autocrine loop. Indeed, neutralizing antibodies against IL6 and IL8 reversed the effects of SCM on MCF-7, pinpointing these cytokines as major mediators of EMT and stemness-related effects associated with the senescent microenvironment. Additionally, prolonged exposure of MCF cells to IL6 or IL8 induced the appearance of senescent cells, suggesting a mechanism by which senescence and inflammation are reinforced favouring the acquisition of EMT and stem-like features at the population level, thus increasing tumour aggressiveness. Strikingly, our results also show that both IL6 and IL8 are important to maintain aggressive traits in MDA-MB-231 cells, a highly tumorigenic cell line, which appears to be devoid of stemness-related features. This study demonstrates that, similar to what is observed with a senescent microenvironment, purified IL6 and IL8 induce a self- and cross-reinforced senescence/inflammatory milieu responsible for the emergence of epithelial plasticity and stemness features, thus conferring more aggressive phenotypes to a luminal breast cancer cell line. On the other hand, the basal-like MDA-MB-231 cells, whose aggressiveness-related features depend on IL6 and IL8 secretion, almost completely lack mammosphere formation and differentiation capacities, suggesting that tumour aggressiveness is not always related to stemness.

MeSH Terms

  • Breast Neoplasms
  • Carcinogenesis
  • Cellular Senescence
  • Culture Media, Conditioned
  • Humans
  • Inflammation
  • Interleukin-6
  • Interleukin-8
  • MCF-7 Cells
  • Phenotype

Keywords

  • Breast cancer
  • IL6
  • IL8
  • Inflammation
  • Senescence
  • Stemness


Sialylation regulates myofibroblast differentiation of human skin fibroblasts.

Fibroblasts are key players in maintaining skin homeostasis and in orchestrating physiological tissue repair and skin regeneration. Dysfunctions in fibroblasts that occur with aging and the senescent process lead to the delayed healing observed in elderly people. The molecular mechanisms leading to fibroblast dysfunction during aging and the senescent process have not yet been clarified. Previously, changes in patterns of glycosylation were observed in fibroblasts in aging and the senescent process, but the effect of these changes on the function of fibroblasts has not been well documented. Here, we investigated whether changes in glycosylation during the process to senescence may have functional effects on fibroblasts. The changes in cell surface glycans on skin fibroblasts during the process to senescence were examined in early-passage (EP) and late-passage (LP) skin fibroblasts by fluorescence-activated cell sorting analysis using lectins. The contributors to the changes in cell surface glycans were examined by real-time polymerase chain reaction or Western blot analysis. The effects of changes in glycosylation on proliferation, migration, induction of cellular senescence, and myofibroblast differentiation induced by transforming growth factor (TGF)-β1 stimulation were examined in EP fibroblasts. The changes in glycosylation were performed by GalNAc-α-O-benzyl or sialidase treatment. A decrease in sialylation of glycoproteins and an increase in sialidase NEU1 were observed in LP fibroblasts. The reduction of sialylation did not have any effect on proliferation, migration, or induction of cellular senescence. On the other hand, myofibroblast differentiation was inhibited by the reduction of sialylation, indicating that sialylation is important for myofibroblast differentiation. The localization of CD44 in lipid rafts, which is required for myofibroblast differentiation, was inhibited by the reduction of sialylation. Furthermore, reduced myofibroblast differentiation in LP fibroblasts was restored by a sialidase inhibitor. Desialylation of CD44 with increased sialidase during the process to senescence reduced the localization of CD44 in lipid rafts after TGF-β1 stimulation, leading to the inhibition of myofibroblast differentiation. Thus, regulation of sialylation may be an attractive strategy for the prevention and regenerative therapy of age-related skin diseases, cosmetic skin alterations, and chronic wounds caused by delayed healing in elderly people.

MeSH Terms

  • Cell Differentiation
  • Cells, Cultured
  • Glycosylation
  • Humans
  • Hyaluronan Receptors
  • Myofibroblasts
  • Polysaccharides
  • Protein Processing, Post-Translational
  • Sialic Acids
  • Skin

Keywords

  • Aging
  • CD44
  • Lipid rafts
  • Myofibroblast differentiation
  • Sialidase
  • Sialylation


Calorie Restriction Attenuates Terminal Differentiation of Immune Cells.

Immune senescence is a natural consequence of aging and may contribute to frailty and loss of homeostasis in later life. Calorie restriction increases healthy life-span in C57BL/6J (but not DBA/2J) mice, but whether this is related to preservation of immune function, and how it interacts with aging, is unclear. We compared phenotypic and functional characteristics of natural killer (NK) cells and T cells, across the lifespan, of calorie-restricted (CR) and control C57BL/6 and DBA/2 mice. Calorie restriction preserves a naïve T cell phenotype and an immature NK cell phenotype as mice age. The splenic T cell populations of CR mice had higher proportions of CD11a CD44 cells, lower expression of TRAIL, KLRG1, and CXCR3, and higher expression of CD127, compared to control mice. Similarly, splenic NK cells from CR mice had higher proportions of less differentiated CD11b CD27 cells and correspondingly lower proportions of highly differentiated CD11b CD27 NK cells. Within each of these subsets, cells from CR mice had higher expression of CD127, CD25, TRAIL, NKG2A/C/E, and CXCR3 and lower expression of KLRG1 and Ly49 receptors compared to controls. The effects of calorie restriction on lymphoid cell populations in lung, liver, and lymph nodes were identical to those seen in the spleen, indicating that this is a system-wide effect. The impact of calorie restriction on NK cell and T cell maturation is much more profound than the effect of aging and, indeed, calorie restriction attenuates these age-associated changes. Importantly, the effects of calorie restriction on lymphocyte maturation were more marked in C57BL/6 than in DBA/2J mice indicating that delayed lymphocyte maturation correlates with extended lifespan. These findings have implications for understanding the interaction between nutritional status, immunity, and healthy lifespan in aging populations.


Keywords

  • T cell
  • aging
  • calorie restriction
  • differentiation
  • maturation
  • natural killer cell


Identification and characterization of adult mouse meniscus stem/progenitor cells.

Meniscal damage is a common problem that accelerates the onset of knee osteoarthritis. Stem cell-based tissue engineering treatment approaches have shown promise in preserving meniscal tissue and restoring meniscal function. The purpose of our study was to identify meniscus-derived stem/progenitor cells (MSPCs) from mouse, a model system that allows for in vivo analysis of the mechanisms underlying meniscal injury and healing. MSPCs were isolated from murine menisci grown in explant culture and characterized for stem cell properties. Flow cytometry was used to detect the presence of surface antigens related to stem cells, and qRT-PCR was used to examine the gene expression profile of MSPCs. Major proteins associated with MSPCs were localized in the adult mouse knee using immunohistochemistry. Our data show that MSPCs have universal stem cell-like properties including clonogenicity and multi-potentiality. MSPCs expressed the mesenchymal stem cell markers CD44, Sca-1, CD90, and CD73 and when cultured had elevated levels of biglycan and collagen type I, important extracellular matrix components of adult meniscus. MSPC also expressed significant levels of Lox and Igf-1, genes associated with the embryonic meniscus. Localization studies showed staining for these same proteins in the superficial and outer zones of the adult mouse meniscus, regions thought to harbor endogenous repair cells. MSPCs represent a novel resident stem cell population in the murine meniscus. Analysis of MSPCs in mice will allow for a greater understanding of the cell biology of the meniscus, essential information for enhancing therapeutic strategies for treating knee joint injury and disease.

MeSH Terms

  • Adult Stem Cells
  • Aging
  • Animals
  • Cell Separation
  • Cells, Cultured
  • Flow Cytometry
  • Gene Expression Profiling
  • Meniscus
  • Mice, Inbred C57BL
  • Stem Cells

Keywords

  • Mouse meniscus
  • osteoarthritis
  • progenitor cells
  • stem cells


Development and mechanistic insight into enhanced cytotoxic potential of hyaluronic acid conjugated nanoparticles in CD44 overexpressing cancer cells.

The overexpression of CD44 in cancer cells reroutes number of oncogenic pathways including the central Pi3K/Akt/NF-kB pathway leading to cancer progression and malignancy. Herein, we developed hyaluronic acid-modified poly(dl-lactic-co-glycolic acid)-poly (ethylene glycol) nanoparticles (PLGA-PEG-HA NPs) for targeted delivery of TTQ (thio-tetrazolyl analog of a clinical candidate, IC87114) to CD44 overexpressing cancer cells. The PLGA-PEG co-polymer was synthesized and characterized by NMR and FTIR. The co-polymer based nanoparticles were prepared by solvent evaporation method and hyaluronic acid (HA) was conjugated on to the nanoparticle surface via EDC/NHS chemistry. The PLGA-PEG-HA NPs had a desirable particle size (<200nm) with reduced polydispersibility and exhibited spherical shape under atomic force microscope (AFM). In vitro cytotoxicity and cellular uptake studies demonstrated higher cytotoxicity and enhanced intracellular accumulation of PLGA-PEG-HA NPs compared to PLGA-PEG NPs in high CD44 expressing MiaPaca-2 cells compared to MDA-MB-231 and MCF7 cells. At the molecular level, the PLGA-PEG-HA NPs were found to be inducing premature senescence with increase in senescence associated β-galactosidase activity and senescence specific marker p21 expression through modulation of Pi3K/Akt/NF-kB signaling pathway in MiaPaca-2 cells. These findings collectively indicated that HA-modified nanoparticles might serve as a promising nanocarrier for site-specific drug delivery, and can be explored further to increase the therapeutic efficacy of anticancer drugs via targeting to CD44 over-expressing cancer cells.

MeSH Terms

  • Antineoplastic Agents
  • Cell Line, Tumor
  • Cell Proliferation
  • Cell Survival
  • Cytotoxins
  • Dose-Response Relationship, Drug
  • Drug Compounding
  • Humans
  • Hyaluronan Receptors
  • Hyaluronic Acid
  • Lactic Acid
  • MCF-7 Cells
  • Nanoparticles
  • Polyglycolic Acid
  • Polylactic Acid-Polyglycolic Acid Copolymer

Keywords

  • CD44 targeted delivery
  • Dicyclohexylcarbodiimide (PubChem CID: 10868)
  • Hyaluronic acid
  • N-hydroxysuccinimide (PubChem CID: 80170)
  • Nanoparticle
  • PLGA
  • Senescence
  • Sodium hyaluronate (PubChem CID: 3084049)
  • Triethylamine (PubChem CID: 8471)
  • poly(dl-lactic-co-glycolic acid) (PubChem CID: 23111554)


Review: Astrocytes in Alzheimer's disease and other age-associated dementias: a supporting player with a central role.

Astrocytes have essential roles in the central nervous system and are also implicated in the pathogenesis of neurodegenerative disease. Forming non-overlapping domains, astrocytes are highly complex cells. Immunohistochemistry to a variety of proteins can be used to study astrocytes in tissue, labelling different cellular components and sub-populations, including glial fibrillary acidic protein, ALDH1L1, CD44, NDRG2 and amino acid transporters, but none of these labels the entire astrocyte population. Increasing heterogeneity is recognized in the astrocyte population, a complexity that is relevant both to their normal function and pathogenic roles. They are involved in neuronal support, as active components of the tripartite synapse and in cell interactions within the neurovascular unit (NVU), where they are essential for blood-brain barrier maintenance and neurovascular coupling. Astrocytes change with age, and their responses may modulate the cellular effects of neurodegenerative pathologies, which alone do not explain all of the variance in statistical models of neurodegenerative dementias. Astrocytes respond to both the neurofibrillary tangles and plaques of Alzheimer's disease, to hyperphosphorylated tau and Aβ, eliciting an effect which may be neuroprotective or deleterious. Not only astrocyte hypertrophy, in the form of gliosis, occurs, but also astrocyte injury and atrophy. Loss of normal astrocyte functions may contribute to reduced support for neurones and dysfunction of the NVU. Understanding how astrocytes contribute to dementia requires an understanding of the underlying heterogeneity of astrocyte populations, and the complexity of their responses to pathology. Enhancing the supportive and neuroprotective components of the astrocyte response has potential translational applications in therapeutic approaches to dementia.

MeSH Terms

  • Aging
  • Alzheimer Disease
  • Animals
  • Astrocytes
  • Dementia
  • Humans

Keywords

  • Alzheimer's disease
  • astrocytes
  • dementia
  • neurodegeneration
  • neurovascular unit


Disentangling the aneuploidy and senescence paradoxes: a study of triploid breast cancers non-responsive to neoadjuvant therapy.

Aneuploid cells should have a reduced proliferation rate due to difficulty in proceeding through mitosis. However, contrary to this, high aneuploidy is associated with aggressive tumour growth and poor survival prognosis, in particular in triploid breast cancer. A further paradox revolves around the observation that, while cell senescence should inhibit proliferation, the senescence marker p16INK4a correlates with poor treatment outcome in patients with a very aggressive triple-negative breast carcinoma (TNBC). In this study, we aim to pour light on the possible relationship of these conundrums with polyploidy of tumour cells. We performed detailed analysis of DNA histogram profiles in diagnostic core biopsies of 30 cases of operable breast cancer and found that near triploidy in TNBC and other forms correlated with weak or no response to neoadjuvant chemotherapy (NAC) as scored by Miller-Payne index. Polyploid cells in operation samples from tumours that were non-responsive to NAC treatment were Ki67 and CD44 positive. In addition, polyploid cells were positive for markers of embryonic stemness (OCT4, SOX2, NANOG) and senescence (p16INK4a). The relationship patterns between p16INK4a and NANOG were heterogeneous, with predominantly mutually exclusive expression but also synergistic and intermediate variants in the same samples. We conclude that the aneuploidy and senescence paradoxes can be explained by the mutual platform of polyploidy, conferring genomic and epigenetic instability as a survival advantage. Such cells are able to bypass aneuploidy restrictions of conventional mitosis and overcome the barrier of senescence by a shift to self-renewal, resulting in progression of cancer.

MeSH Terms

  • Adult
  • Aged
  • Aneuploidy
  • Breast Neoplasms
  • Cellular Senescence
  • DNA, Neoplasm
  • Female
  • Humans
  • Middle Aged
  • Neoadjuvant Therapy
  • Triploidy

Keywords

  • Breast cancer
  • Resistance
  • Self-renewal
  • Senescence
  • Triple-negative
  • Triploidy


Heightened self-reactivity associated with selective survival, but not expansion, of naïve virus-specific CD8 T cells in aged mice.

In advanced age, decreased CD8( ) cytotoxic T-lymphocyte (CTL) responses to novel pathogens and cancer is paralleled by a decline in the number and function of naïve CTL precursors (CTLp). Although the age-related fall in CD8( ) T-cell numbers is well established, neither the underlying mechanisms nor the extent of variation for different epitope specificities have been defined. Furthermore, naïve CD8( ) T cells expressing high levels of CD44 accumulate with age, but it is unknown whether this accumulation reflects their preferential survival or an age-dependent driver of CD8( ) T-cell proliferation. Here, we track the number and phenotype of four influenza A virus (IAV)-specific CTLp populations in naïve C57BL/6 (B6) mice during aging, and compare T-cell receptor (TCR) clonal diversity for the CD44hi and CD44lo subsets of one such population. We show differential onset of decline for several IAV-specific CD8( ) T-cell populations with advanced age that parallel age-associated changes in the B6 immunodominance hierarchy, suggestive of distinct impacts of aging on different epitope-specific populations. Despite finding no evidence of clonal expansions in an aged, epitope-specific TCR repertoire, nonrandom alterations in TCR usage were observed, along with elevated CD5 and CD8 coreceptor expression. Collectively, these data demonstrate that naïve CD8( ) T cells expressing markers of heightened self-recognition are selectively retained, but not clonally expanded, during aging.

MeSH Terms

  • Animals
  • CD8-Positive T-Lymphocytes
  • Mice
  • Mice, Inbred C57BL

Keywords

  • CD44 virtual memory
  • CD5
  • aging
  • influenza A virus
  • naive CD8 T cells


Characterization of age-associated exhausted CD8⁺ T cells defined by increased expression of Tim-3 and PD-1.

Aging is accompanied by altered T-cell responses that result in susceptibility to various diseases. Previous findings on the increased expression of inhibitory receptors, such as programmed cell death protein 1 (PD-1), in the T cells of aged mice emphasize the importance of investigations into the relationship between T-cell exhaustion and aging-associated immune dysfunction. In this study, we demonstrate that T-cell immunoglobulin mucin domain-3 (Tim-3), another exhaustion marker, is up-regulated on aged T cells, especially CD8( ) T cells. Tim-3-expressing cells also produced PD-1, but Tim-3( ) PD-1( ) CD8( ) T cells had a distinct phenotype that included the expression of CD44 and CD62L, from Tim-3(-) PD-1( ) cells. Tim-3( ) PD-1( ) CD8( ) T cells showed more evident properties associated with exhaustion than Tim-3(-) PD-1( ) CD8( ) T cells: an exhaustion-related marker expression profile, proliferative defects following homeostatic or TCR stimulation, and altered production of cytokines. Interestingly, these cells produced a high level of IL-10 and induced normal CD8( ) T cells to produce IL-10, which might contribute to immune dysregulation in aged mice. The generation of Tim-3-expressing CD8( ) T cells in aged mice seems to be mediated by encounters with antigens but not by specific infection, based on their high expression of CD49d and their unbiased TCR Vβ usage. In conclusion, we found that a CD8( ) T-cell population with age-associated exhaustion was distinguishable by its expression of Tim-3. These results provide clues for understanding the alterations that occur in T-cell populations with age and for improving dysfunctions related to the aging of the immune system.

MeSH Terms

  • Aging
  • Animals
  • CD8-Positive T-Lymphocytes
  • Hepatitis A Virus Cellular Receptor 2
  • Mice
  • Programmed Cell Death 1 Receptor

Keywords

  • Aging
  • CD8 T cells
  • PD-1
  • T-cell exhaustion
  • Tim-3


miR-34a inhibits differentiation of human adipose tissue-derived stem cells by regulating cell cycle and senescence induction.

MicroRNAs (miRNAs) are critical in the maintenance, differentiation, and lineage commitment of stem cells. Stem cells have the unique property to differentiate into tissue-specific cell types (lineage commitment) during cell division (self-renewal). In this study, we investigated whether miR-34a, a cell cycle-regulating microRNA, could control the stem cell properties of adipose tissue-derived stem cells (ADSCs). First, we found that the expression level of miR-34a was increased as the cell passage number was increased. This finding, however, was inversely correlated with our finding that the overexpression of miR-34a induced the decrease of cell proliferation. In addition, miR-34a overexpression decreased the expression of various cell cycle regulators such as CDKs (-2, -4, -6) and cyclins (-E, -D), but not p21 and p53. The cell cycle analysis showed accumulation of dividing cells at S phase by miR-34a, which was reversible by co-treatment with anti-miR-34a. The potential of adipogenesis and osteogenesis of ADSCs was also decreased by miR-34a overexpression, which was recovered by co-treatment with anti-miR-34a. The surface expression of stem cell markers including CD44 was also down-regulated by miR-34a overexpression as similar to that elicited by cell cycle inhibitors. miR-34a also caused a significant decrease in mRNA expression of stem cell transcription factors as well as STAT-3 expression and phosphorylation. Cytokine profiling revealed that miR-34a significantly modulated IL-6 and -8 production, which was strongly related to cellular senescence. These data suggest the importance of miR-34a for the fate of ADSCs toward senescence rather than differentiation.

MeSH Terms

  • Adipogenesis
  • Adipose Tissue
  • Cell Cycle
  • Cell Cycle Proteins
  • Cell Proliferation
  • Cellular Senescence
  • Humans
  • Hyaluronan Receptors
  • Interleukin-6
  • Interleukin-8
  • MicroRNAs
  • Oligoribonucleotides, Antisense
  • Osteogenesis
  • STAT3 Transcription Factor
  • Stem Cells

Keywords

  • Adipose-derived Mesenchymal Stem Cells
  • Cell Cycle
  • Senescence
  • miR-34a


Microneedle fractional radiofrequency increases epidermal hyaluronan and reverses age-related epidermal dysfunction.

Skin aging results in physiological alterations in keratinocyte activities and epidermal function, as well as dermal changes. Yet, the cellular and molecular mechanisms that cause epidermal dysfunction during skin aging are not well understood. Recently, the role of epidermal hyaluronan (HA) as an active regulator of dynamic cellular processes is getting attention and alterations in HA metabolism are thought to be important in age-related epidermal dysfunction. Microneedle fractional radiofrequency (RF) has shown effects for improving cutaneous aging. However, little is known about the effects of fractional RF on the epidermal HA and epidermal function. We investigated the effect of microneedle fractional RF on the expression of epidermal HA in young and aged mice epidermis. We performed fractional RF on the dorsal skin of 30 8-week-old (young) hairless mice and 15 47-week-old (aged) C57BL/6J mice. Skin samples were collected on day 1, 3, and 7. HA content was measured by ELISA. Gene expressions of CD 44, HABP4, and HAS3 were measured using real time RT-PCR. Immunohistochemistry for detection of HA, CD44, PCNA, and filaggrin were performed. HA content and the mRNA levels of HABP4, CD44, and HAS3 were upregulated in the epidermis of both young and aged mice after microneedle fractional RF treatment. The expression was increased from day 1 after treatment and increased expression persisted on day 7. Fractional RF treatment significantly increased PCNA and filaggrin expression only in the aged mice skin. Microneedle fractional RF increased epidermal HA and CD44 expression in both young and aged mice and reversed age-related epidermal dysfunction especially in aged mice, suggesting a new mechanism involved in the skin rejuvenation effect of microneedle fractional RF.

MeSH Terms

  • Animals
  • Biomarkers
  • Cell Proliferation
  • Epidermis
  • Female
  • Homeostasis
  • Hyaluronic Acid
  • Mice
  • Mice, Inbred C57BL
  • Needles
  • Radio Waves
  • Skin Aging

Keywords

  • CD44
  • PCNA
  • aging
  • filaggrin
  • hyaluronan
  • hyaluronan synthase
  • radiofrequency


Age-independent effects of hyaluronan amide derivative and growth hormone on human osteoarthritic chondrocytes.

Increased age is the most prominent risk factor for the initiation and progression of osteoarthritis (OA). The effects of human growth hormone (hGH) combined or not with hyaluronan amide derivative (HAD) were evaluated on human OA chondrocytes, to define their biological action and potentiality in OA treatment. Cell viability, metabolic activity, gene expression and factors released were tested at different time points on chondrocytes treated with different concentrations of hGH (0.01-10 μg/ml) alone or in combination with HAD (1 mg/ml). We found that OA chondrocytes express GH receptor and that the different doses of hGH tested did not affect cell viability, metabolic activity or the expression of collagen type 2, 1, or 10 nor did it induce the release of IGF-1 or FGF-2. Conversely, hGH treatment increased the expression of hyaluronan receptor CD44. HAD combined with hGH reduced metabolic activity, IL6 release and gene expression, but not the suppressor of cytokine signaling 2 (SOCS2), which was significantly induced and translocated into the nucleus. The parameters analyzed, independently of the treatments used proportionally decreased with increasing age of the patients. hGH only induced CD44 receptor on OA chondrocytes but did not affect other parameters, such as chondrocytic gene markers or IGF-1 or FGF-2 release. HAD reduced all the effects induced by hGH partially through a significant induction of SOCS2. These data show that GH or HAD treatment does not influence the response of the OA chondrocytes, thus the modulation of cellular response is age-independent.

MeSH Terms

  • Aged
  • Aging
  • Cells, Cultured
  • Chondrocytes
  • Female
  • Human Growth Hormone
  • Humans
  • Hyaluronic Acid
  • Male
  • Middle Aged
  • Osteoarthritis, Hip

Keywords

  • Age
  • chondrocytes
  • growth hormone
  • hyaluronan
  • osteoarthritis


Changes in glomerular parietal epithelial cells in mouse kidneys with advanced age.

Kidney aging is accompanied by characteristic changes in the glomerulus, but little is known about the effect of aging on glomerular parietal epithelial cells (PECs), nor if the characteristic glomerular changes in humans and rats also occur in very old mice. Accordingly, a descriptive analysis was undertaken in 27-mo-old C57B6 mice, considered advanced age. PEC density was significantly lower in older mice compared with young mice (aged 3 mo), and the decrease was more pronounced in juxtamedullary glomeruli compared with outer cortical glomeruli. In addition to segmental and global glomerulosclerosis in older mice, staining for matrix proteins collagen type IV and heparan sulfate proteoglycan were markedly increased in Bowman's capsules of older mouse glomeruli, consistent with increased extracellular matrix production by PECs. De novo staining for CD44, a marker of activated and profibrotic PECs, was significantly increased in aged glomeruli. CD44 staining was more pronounced in the juxtamedullary region and colocalized with phosphorylated ERK. Additionally, a subset of aged PECs de novo expressed the epithelial-to-mesenchymal transition markers α-smooth muscle and vimentin, with no changes in epithelial-to-mesenchymal transition markers E-cadherin and β-catenin. The mural cell markers neural/glial antigen 2, PDGF receptor-β, and CD146 as well as Notch 3 were also substantially increased in aged PECs. These data show that mice can be used to better understand the aging kidney and that PECs undergo substantial changes, especially in juxtamedullary glomeruli, that may participate in the overall decline in glomerular structure and function with advancing age.

MeSH Terms

  • Aging
  • Animals
  • Biomarkers
  • Bowman Capsule
  • Epithelial Cells
  • Epithelial-Mesenchymal Transition
  • Extracellular Matrix Proteins
  • Extracellular Signal-Regulated MAP Kinases
  • Female
  • Hyaluronan Receptors
  • Kidney Glomerulus
  • Mice, Inbred C57BL
  • Pericytes
  • Phosphorylation
  • Podocytes
  • Receptor, Notch3
  • Receptors, Notch

Keywords

  • CD146
  • CD44
  • Notch 3
  • collagen type IV
  • epithelial-to-mesenchymal transition
  • extracellular signal-regulated kinase
  • glomerulosclerosis
  • glomerulus
  • heparan sulfate proteoglycan
  • neural/glial antigen 2
  • platelet-derived growth factor receptor-β
  • podocyte
  • vimentin
  • α-smooth muscle actin


In vitro 3-D model based on extending time of culture for studying chronological epidermis aging.

Skin aging is a complex phenomenon in which several mechanisms operate simultaneously. Among them, intrinsic aging is a time-dependent process, which leads to gradual skin changes affecting its structure and function such as thinning down of both epidermal and dermal compartments and a flattening and fragility of the dermo-epidermal junction. Today, several approaches have been proposed for the generation of aged skin in vitro, including skin explants from aged donors and three-dimensional skin equivalent treated by aging-inducing chemical compounds or engineered with human cells isolated from aged donors. The aim of this study was to develop and validate a new in vitro model of aging based on skin equivalent demonstrating the same phenotypic changes that were observed in chronological aging. By using prolonged culture as a proxy for cellular aging, we extended to 120 days the culture time of a skin equivalent model based on collagen-glycosaminoglycan-chitosan porous polymer and engineered with human skin cells from photo-protected sites of young donors. Morphological, immunohistological and ultrastructural analysis at different time points of the culture allowed characterizing the phenotypic changes observed in our model in comparison to samples of non photo-exposed normal human skin from different ages. We firstly confirmed that long-term cultured skin equivalents are still morphologically consistent and functionally active even after 120 days of culture. However, similar to in vivo chronological skin aging a significant decrease of the epidermis thickness as well as the number of keratinocyte expressing proliferation marker Ki67 are observed in extended culture time skin equivalent. Epidermal differentiation markers loricrin, filaggrin, involucrin and transglutaminase, also strongly decreased. Ultrastructural analysis of basement membrane showed typical features of aged skin such as duplication of lamina densa and alterations of hemidesmosomes. Moreover, the expression of hyaluronan and its surface receptor CD44 drastically decreased as observed during chronological skin aging. Finally, we found that the level of p16INK4A expression significantly increased supporting cellular senescence process associated to our model. To conclude, the major morphological and ultrastructural epidermal modifications observed in both our extended culture skin equivalent model and skin biopsies from old donors validate the relevance of our model for studying chronological aging, understanding and elucidating age-related modifications of basic skin biological processes. In addition, our model provides a unique tool for identifying new targeted molecules intended at improving the appearance of aging skin.

MeSH Terms

  • Adult
  • Aged
  • Aging
  • Basement Membrane
  • Cellular Senescence
  • Epidermis
  • Extracellular Matrix
  • Humans
  • Hyaluronan Receptors
  • Hyaluronic Acid
  • Ki-67 Antigen
  • Middle Aged
  • Models, Biological
  • Tissue Culture Techniques
  • Young Adult

Keywords

  • Aging
  • Epidermis
  • Extended culture time
  • Senescence
  • Skin equivalent model


Premature aging induced by radiation exhibits pro-atherosclerotic effects mediated by epigenetic activation of CD44 expression.

Age is undoubtedly a major risk factor for heart disease. However, the reason for this is not entirely clear. In the course of our investigation into the mechanism of radiation-induced cardiovascular disease, we made several unexpected findings that inform us on this question. We observed that human coronary endothelial cells, while being able to initiate repair of radiation-induced DNA damage, often fail to complete the repair and become senescent. Such radiation-induced cellular aging occurs through a mutation-independent route. Endothelial cells that aged naturally through replication or as a result of radiation exhibited indistinguishable characteristics. The promoter regions of the CD44 gene in aging endothelial cells become demethylated, and the proteins are highly expressed on the cell surface, making the cells adhesive for monocytes. Adhesion is a cardinal feature that recruits monocytes to the endothelium, allowing them to infiltrate the vessel wall and initiate atherosclerosis. The epigenetic activation of CD44 expression is particularly significant as it causes persistent elevated CD44 protein expression, making senescent endothelial cells chronically adhesive. In addition to understanding why cardiovascular disease increases with age, these observations provide insights into the puzzling association between radiation and cardiovascular disease and highlight the need to consider premature aging as an additional risk of radiation to human health.

MeSH Terms

  • Aging, Premature
  • Animals
  • Atherosclerosis
  • Cell Adhesion
  • Coronary Artery Disease
  • Coronary Vessels
  • Endothelium, Vascular
  • Epigenesis, Genetic
  • HL-60 Cells
  • Humans
  • Hyaluronan Receptors
  • Monocytes
  • Radiation Injuries, Experimental
  • Risk Factors

Keywords

  • CD44
  • endothelium
  • inflammation
  • irradiation
  • monocyte adhesion
  • senescence


Hyaluronan enhances wound repair and increases collagen III in aged dermal wounds.

Age-related changes in the extracellular matrix contribute to delayed wound repair in aging. Hyaluronan, a linear nonsulfated glycosaminoglycan, promotes synthesis and assembly of key extracellular matrix components, such as the interstitial collagens, during wound healing. The biological effects of hyaluronan are mediated, in part, by hyaluronan size. We have previously determined that dermal wounds in aged mice, relative to young mice, have deficits in the generation of lower molecular weight hyaluronan (defined as <300 kDa). Here, we tested the effect of exogenous hyaluronan of 2, 250, or 1,000 kDa sizes on full-thickness excisional wounds in aged mice. Only wounds treated with 250 kDa hyaluronan (HA250) were significantly improved over wounds that received carrier (water) alone. Treatment with HA250 was associated with increased expression of transcripts for the hyaluronan receptors CD44 and RHAMM, as well as collagens III and I. Analyses of dermal protein content by mass spectrometry and Western blotting confirmed significantly increased expression of collagen III in wounds treated with HA250 relative to control wounds. In summary, we find that HA250 improves wound repair and increases the synthesis of collagen III in aged dermal wounds.

MeSH Terms

  • Aging
  • Animals
  • Blotting, Western
  • Collagen Type III
  • Dermis
  • Extracellular Matrix
  • Extracellular Matrix Proteins
  • Hyaluronan Receptors
  • Hyaluronic Acid
  • Male
  • Mass Spectrometry
  • Mice
  • Mice, Inbred C57BL
  • RNA, Messenger
  • Soft Tissue Injuries
  • Wound Healing


Metzincins and related genes in experimental renal ageing: towards a unifying fibrosis classifier across species.

We have previously described a transcriptomic classifier consisting of metzincins and related genes (MARGS) discriminating kidneys and other organs with or without fibrosis from human biopsies. We now apply our MARGS-based algorithm to a rat model of age-associated interstitial renal fibrosis. Untreated Fisher 344 rats (n = 76) were sacrificed between 2 to 104 weeks of age. For gene expression studies, we used single colour (Cy3) Agilent Whole Rat Genome 4 × 44k microarrays; 4-5 animals of each sex were profiled at each of the following ages: 2, 5, 6, 8, 15, 21, 78 and 104 weeks. Intensity data were subjected to variance stabilization (www.Partek.com). Data were analysed with ANOVA and other statistical methods. Sixty MARGS were differentially expressed across age groups. More MARGS were differentially expressed in older males than in older females. Principal component analysis showed gene expression induced segregation of age groups by sex from 6 to 104 weeks of age. The expression level of MMP7 correlated best with fibrosis grade. Severity of fibrosis was determined in 20 animals at 78 and 104 weeks of age. Expression values of 15 of 19 genes of the original classifier present on the Agilent array, in conjunction with linear discriminant analysis, was sufficient to correctly classify these 20 samples into non-fibrosis and fibrosis. Overrepresentation of MMP2 protein and CD44 protein in fibrosis was confirmed by immunofluorescence. Based on these results and our previous work, the MARGS classifier represents a cross-organ and cross-species classifier of fibrosis irrespective of aetiology. This finding provides evidence for a common pathway leading to fibrosis and will help to design a PCR-based clinical test.

MeSH Terms

  • Aging
  • Algorithms
  • Animals
  • Disease Models, Animal
  • Female
  • Fibrosis
  • Gene Expression Profiling
  • Kidney
  • Male
  • Matrix Metalloproteinase 2
  • Matrix Metalloproteinase 7
  • Microarray Analysis
  • Polymerase Chain Reaction
  • Principal Component Analysis
  • Rats
  • Rats, Inbred F344
  • Transcriptome

Keywords

  • CD44
  • ageing
  • classifier
  • fibrosis
  • matrix metalloproteinases
  • metzincins


Immunohistochemical patterns in the interfollicular Caucasian scalps: influences of age, gender, and alopecia.

Skin ageing and gender influences on the scalp have been seldom studied. We revisited the changes in the interfollicular scalp. The study was performed on a population of 650 volunteers (300 women and 350 men) for over 7 years. Three age groups were selected in both genders, namely, subjects aged 20-35, 50-60, and 60-70 years. The hair status was further considered according to nonalopecic and alopecic patterns and severity (discrete, moderate, and severe). Biopsies from the parietal area were processed for immunohistochemistry. Stromal cells were distinguished according to the presence of vimentin, Factor XIIIa, CD117, and versican. Blood and lymphatic vessels were highlighted by Ulex europaeus agglutinin-1 and human podoplanin immunoreactivities, respectively. Actinic elastosis was identified by the lysozyme coating of elastic fibres. The epidermis was explored using the CD44 variant 3 and Ki67 immunolabellings. Biplot analyses were performed. Immunohistochemistry revealed a prominent gender effect in young adults. Both Factor XIIIa dermal dendrocytes and the microvasculature size decreased with scalp ageing. Alopecia changes mimicked stress-induced premature senescence.

MeSH Terms

  • Adult
  • Aged
  • Aging
  • Alopecia
  • Biopsy
  • Epidermis
  • European Continental Ancestry Group
  • Factor XIIIa
  • Female
  • Humans
  • Male
  • Middle Aged
  • Proto-Oncogene Proteins c-kit
  • Scalp
  • Sex Characteristics
  • Skin Aging
  • Versicans
  • Vimentin


Human leukemic cell lines synthesize hyaluronan to avoid senescence and resist chemotherapy.

Hyaluronan (HA) is one of the major components of the extracellular matrix. Several solid tumors produce high levels of HA, which promotes survival and multidrug resistance (MDR). HA oligomers (oHAs) can block HA effects. However, little is known about the role of HA in hematological malignancies. The aim of this work was to determine whether HA or its oligomers can modulate the proliferation of leukemia cells as well as their effect on MDR. Receptors and signaling pathways involved were also analyzed. For this purpose, the human leukemic cell lines K562 and Kv562, which are sensitive and resistant to Vincristine (VCR), respectively, were used. We demonstrated that HA induced cell proliferation in both cell lines. On K562 cells, this effect was mediated by cluster differentiation 44 (CD44) and activation of both phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways, whereas on Kv562 cells, the effect was mediated by receptor for hyaluronan-mediated motility (RHAMM) and PI3K/Akt activation. The inhibition of HA synthesis by 4-methylumbelliferone (4MU) decreased cell line proliferation and sensitized Kv562 to the effect of VCR through P-glycoprotein (Pgp) inhibition, in both cases with senescence induction. Moreover, oHAs inhibited K562 proliferation mediated by CD44 as well as Akt and ERK down-regulation. Furthermore, oHAs sensitized Kv562 cells to VCR by Pgp inhibition inducing senescence. We postulate that the synthesis of HA would promote leukemia progression mediated by the triggering of the above-mentioned proliferative signals. These findings highlight the potential use of oHAs and 4MU as coadjuvant for drug-resistant leukemia.

MeSH Terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Cell Proliferation
  • Cell Survival
  • Cellular Senescence
  • Dose-Response Relationship, Drug
  • Drug Resistance, Multiple
  • Drug Resistance, Neoplasm
  • Humans
  • Hyaluronic Acid
  • Hymecromone
  • K562 Cells
  • Leukemia
  • Structure-Activity Relationship
  • Tumor Cells, Cultured
  • Vincristine

Keywords

  • CD44
  • RHAMM
  • drug resistance
  • hyaluronan
  • leukemia
  • senescence


Mice with heterozygous deficiency of manganese superoxide dismutase (SOD2) have a skin immune system with features of "inflamm-aging".

Dendritic cells (DC) are central in regulating skin immunity. Immunosenescence is associated with a chronic inflammatory state. Little is known about the contribution of DC to "inflamm-aging". When determining langerhans cell (LC) numbers, we found a 60 % reduction of LC in aged epidermis. Reactive oxygen species(ROS) are linked with aging. The mitochondrial manganese superoxide dismutase (SOD2) is in the first line of antioxidant defense. We investigated the function of DC from SOD2 heterozygous mice (SOD2 /-) and found that at 4 months of age LC numbers are not altered, but activated LC have impaired expression of MHC-II and CD44. Immature SOD2 /- DC produced increased proinflammatory IL-6 and chemokines CXCL1 and CXCL2. Upon challenge SOD2 /- DC accumulated ROS. When activating SOD2 /- DC by LPS they less efficiently upregulated MHC-II, CD86 and CD44. Surprisingly, in vivo contact hypersensitivity (CHS) was enhanced in SOD2 /- mice although SOD2 /- DC were less potent in stimulating wt T cells. However, SOD2 /- T cells showed increased proliferation, even when stimulated with SOD2 /- DC, possibly explaining the increased CHS. Our findings suggest that SOD2 is a molecular candidate in the regulation of "inflamm-aging" conveying both immunosuppressive and proinflammatory signals through alteration of DC and T cell functions.

MeSH Terms

  • Adult
  • Aged
  • Aged, 80 and over
  • Aging
  • Animals
  • B7-2 Antigen
  • Cell Differentiation
  • Cells, Cultured
  • Chemokine CXCL1
  • Chemokine CXCL2
  • Dendritic Cells
  • Dermatitis, Contact
  • Heterozygote
  • Histocompatibility Antigens Class II
  • Humans
  • Hyaluronan Receptors
  • Inflammation
  • Interleukin-6
  • Lymphocyte Activation
  • Mice
  • Mice, Inbred C57BL
  • Mice, Mutant Strains
  • Reactive Oxygen Species
  • Superoxide Dismutase
  • T-Lymphocytes
  • Young Adult


Selective matrix (hyaluronan) interaction with CD44 and RhoGTPase signaling promotes keratinocyte functions and overcomes age-related epidermal dysfunction.

Mouse epidermal chronologic aging is closely associated with aberrant matrix (hyaluronan, HA)-size distribution/production and impaired keratinocyte proliferation/differentiation, leading to a marked thinning of the epidermis with functional consequence that causes a slower recovery of permeability barrier function. The goal of this study is to demonstrate mechanism-based, corrective therapeutic strategies using topical applications of small HA (HAS) and/or large HA (HAL) [or a sequential small HA (HAS) and large HA(HAL) (HAs→HAL) treatment] as well as RhoGTPase signaling perturbation agents to regulate HA/CD44-mediated signaling, thereby restoring normal epidermal function, and permeability barrier homeostasis in aged mouse skin. A number of biochemical, cell biological/molecular, pharmacological and physiological approaches were used to investigate matrix HA-CD44-mediated RhoGTPase signaling in regulating epidermal functions and skin aging. In this study we demonstrated that topical application of small HA (HAS) promotes keratinocyte proliferation and increases skin thickness, while it fails to upregulate keratinocyte differentiation or permeability barrier repair in aged mouse skin. In contrast, large HA (HAL) induces only minimal changes in keratinocyte proliferation and skin thickness, but restores keratinocyte differentiation and improves permeability barrier function in aged epidermis. Since neither HAS nor HAL corrects these epidermal defects in aged CD44 knock-out mice, CD44 likely mediates HA-associated epidermal functions in aged mouse skin. Finally, blockade of Rho-kinase activity with Y27632 or protein kinase-Nγ activity with Ro31-8220 significantly decreased the HA (HAS or HAL)-mediated changes in epidermal function in aged mouse skin. The results of our study show first that HA application of different sizes regulates epidermal proliferation, differentiation and barrier function in aged mouse skin. Second, manipulation of matrix (HA) interaction with CD44 and RhoGTPase signaling could provide further novel therapeutic approaches that could be targeted for the treatment of various aging-related skin disorders.

MeSH Terms

  • Animals
  • Cell Differentiation
  • Cell Proliferation
  • Cells, Cultured
  • Enzyme Inhibitors
  • Epidermis
  • Extracellular Matrix
  • Humans
  • Hyaluronan Receptors
  • Hyaluronic Acid
  • Intermediate Filament Proteins
  • Keratinocytes
  • Male
  • Mice
  • Mice, Knockout
  • Molecular Weight
  • Protein Kinase C
  • Protein Precursors
  • Signal Transduction
  • Skin Aging
  • rho GTP-Binding Proteins
  • rho-Associated Kinases

Keywords

  • CD44
  • Keratinocyte functions
  • Matrix hyaluronan
  • RhoGTPase signaling
  • Skin aging


Intrinsic defects in CD8 T cells with aging contribute to impaired primary antiviral responses.

Aging is associated with altered immune responses, particularly with a diminished CD8 T cell response. Although both intrinsic and extrinsic factors are hypothesized to impact this decreased T cell response, the direct evidence of an intrinsic deficiency in virus-specific CD8 T cells is limited. In this study, a TCR transgenic (Tg) P14 mouse model was utilized to compare the activation and proliferation of the Tg CD8 T cells of young and aged P14 mice upon stimulation with antigen or infection with virus. The proliferation of purified Tg CD8 T cells of aged mice was significantly lower than that of young mice when cultured in vitro with both the LCMV specific peptide and antigen presenting cells from young wild type mice. In addition, expression of the activation markers, CD69, CD25, and CD44, was delayed on Tg T cells of aged mice after stimulation. Importantly, while adoptive transfer of purified Tg CD8 T cells of young or aged mice into young wild type mice resulted in expansion of the Tg CD8 T cells of both ages after LCMV infection, the expansion of the Tg T cells from aged mice was significantly decreased compared with that of the Tg T cells from young mice. However, while the number of IFN-γ secreting Tg CD8 T cells from aged mice was significantly decreased compared to that of young mice, the percentages of Tg CD8 T cells producing IFN-γ were similar in young and aged mice, demonstrating that proliferation, but not function, of the Tg CD8 T cells of aged mice was impaired. Importantly, chronological age alone was not sufficient to predict an altered proliferative response; rather, expression of high levels of CD44 on CD8 T cells of aged mice reflected a decreased proliferative response. These results reveal that alterations intrinsic to CD8 T cells can contribute to the age-associated defects in the primary CD8 T cell response during viral infection.

MeSH Terms

  • Adoptive Transfer
  • Aging
  • Animals
  • CD8-Positive T-Lymphocytes
  • Cell Proliferation
  • Cells, Cultured
  • Disease Models, Animal
  • Immunity, Cellular
  • Immunity, Innate
  • Lymphocytic Choriomeningitis
  • Lymphocytic choriomeningitis virus
  • Mice
  • Mice, Transgenic
  • Spleen


Rejuvenation of aged pig facial skin by transplanting allogeneic granulocyte colony-stimulating factor-induced peripheral blood stem cells from a young pig.

Following a stroke, the administration of stem cells that have been treated with granulocyte colony-stimulating factor (GCSF) can ameliorate functional deficits in both rats and humans. It is not known, however, whether the application of GCSF-mobilized peripheral blood stem cells (PBSCs) to human skin can function as an antiaging treatment. We used a Lanyu pig (Sus scrofa) model, since compared with rodents, the structure of a pig's skin is very similar to human skin, to provide preliminary data on whether these cells can exert antiaging effects over a short time frame. GCSF-mobilized PBSCs from a young male Lanyu pig (5 months) were injected intradermally into the cheek skin of aged female Lanyu pigs, and tissues before and after the cell injections were compared to determine whether this treatment caused skin rejuvenation. Increased levels of collagen, elastin, hyaluronic acid, and the hyaluronic acid receptor CD44 were observed in both dermal and subcutaneous layers following the injection of PBSCs. In addition, the treated skin tissue was tighter and more elastic than adjacent control regions of aged skin tissue. In the epidermal layer, PBSC injection altered the levels of both involucrin and integrin, indicating an increased rate of epidermal cell renewal as evidenced by reductions in both cornified cells and cells of the spinous layers and increases in the number of dividing cells within the basal layer. We found that the exogenous PBSCs, visualized using fluorescence in situ hybridization, were located primarily in hair follicles and adjacent tissues. In summary, PBSC injection restored young skin properties in the skin of aged (90 months) pigs. On the basis of our preliminary data, we conclude that intradermal injection of GCSF-mobilized PBSCs from a young pig can rejuvenate the skin in aged pigs.

MeSH Terms

  • Aging
  • Animals
  • Face
  • Female
  • Granulocyte Colony-Stimulating Factor
  • Humans
  • Male
  • Peripheral Blood Stem Cell Transplantation
  • Rats
  • Rejuvenation
  • Skin
  • Skin Aging
  • Subcutaneous Tissue
  • Sus scrofa
  • Transplantation, Homologous