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P A Y W A L L E D S O U R C E : Mechanisms of Ageing and Development
Highlights
• tBHP treatment induces premature cellular senescence in skin fibroblasts.
• Oxidative stress-induced mitochondrial damage is a characteristic of tBHP-induced senescence.
• In 3D reconstructed skin, tBHP treatment induces epidermal thinning.
• tBHP treatment of ex vivo skin results in a reduction of collagen fiber thickness.
Abstract
Accumulation of senescent cells promotes the development of age-related pathologies and deterioration. In human skin, senescent cells potentially impair structure and function by secreting a mixture of signaling molecules and proteases that influence neighboring cells and degrade extracellular matrix components, such as elastin and collagen. One of the key underlying mechanisms of senescence and extrinsic skin aging is the increase of intracellular reactive oxygen species and resulting oxidative stress.
Tert-butyl hydroperoxide (tBHP) is a known inducer of oxidative stress and cellular damage, acting at least in part by depleting the antioxidant glutathione. Here, we provide a detailed characterization of tBHP-induced senescence in human dermal fibroblasts in monolayer culture. In addition, results obtained with more physiological experimental models revealed that tBHP treated 3D reconstructed skin and ex vivo skin developed signs of chronic tissue damage, displaying reduced epidermal thickness and collagen fiber thinning.
We, therefore, propose that tBHP treatment can be used as a model to study the effects of extrinsic skin aging, focusing mainly on the influence of environmental pollution.
Abbreviations
cPDL: cumulative Population Doublings
D1: Day One
DAPI4′: 6-diamidino-2-phenylindole
ΔΨM: Mitochondrial Membrane Potential
DHE: Dihydroethidium
DMEM: Dulbecco’s Modified Eagle’s Medium
DPBS: Dulbecco's Phosphate Buffered Saline
EDTA: Ethylenediaminetetraacetic acid
FCCP: Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone
GAD_Disease 'Aging': Genetic Association Database Correlated with Aging
GAPDH: Glyceraldehyde 3-phosphate dehydrogenase
H&E: Hematoxylin and Eosin Staining
HFF-1: Human Foreskin Fibroblasts
HSDF: Human Skin Dermal Fibroblasts
HSEK: Human Skin Epidermal Keratinocytes
MMPs: Matrix metalloproteases
mtDNA: mitochondrial DNA
nDNA: nuclear DNA
PBS: Phosphate Buffered Saline
RIPA: Radioimmunoprecipitation Assay
RNAseq: RNA Sequencing
ROS: Reactive Oxygen Species
SASP: Senescence-associated Secretory Phenotype
SA-β-galactosidaseSenescence-associated β-galactosidase
SD: Standard Deviation
tBHP: tert-butyl hydroperoxide
Outline
1. Introduction
2. Material and Methods
2.1. Cell Culture
2.2. Induction of Stress-induced Premature Senescence using tBHP
2.3. Counting Cells and Estimation of Cumulative Population Doublings (cPDL)
2.4. RNA Isolation, cDNA Synthesis and RT-qPCR
2.5. Protein Isolation
2.6. Westernblot
2.7. Cytochemistry for SA-β-galactosidase
2.8. Determination of Apoptotic and Necrotic Cell Death by flow cytometry Using AnnexinV/PI Staining
2.9. Measurement of Cell Surface Area
2.10. γH2AX Immunofluorescence
2.11. Determination of Intracellular ROS Levels by DHE (Dihydroethidium) Staining
2.12. Determination of Mitochondrial ROS Levels by CM-H2XRos Staining
2.13. Determination of Mitochondrial Membrane Potential by JC-1 Staining
2.14. Relative mitochondrial DNA (mtDNA) / nuclear DNA (nDNA) ratio
2.15. RNAseq Analysis
2.16. Production of 3D Reconstructed Skin (Skin Equivalents)
2.17. Preparation of Skin Biopsies
2.18. Treatment of 3D Skin Equivalents and Skin Biopsies with tBHP
2.19. Processing for Histology of 3D Reconstructed Skin and Skin Biopsies
2.20. Measurement of Epidermal Thickness
2.21. RNA Isolation, cDNA synthesis and RT-qPCR from Skin Biopsies
2.22. Collagen Fiber Density Measurement
2.23. Statistical analysis
3. Results
3.1. tBHP treatment induces stress-induced premature senescence in skin fibroblasts
3.2. Oxidative stress-induced DNA and mitochondrial damage are underlying factors of tBHP-induced senescence
3.3. tBHP-induced senescence is connected to tobacco smoke induced pathologies and aging
3.4. tBHP treatment of 3D reconstructed skin equivalents reduces epidermal thickness and increases epidermal thickness variance in ex vivo skin
3.5. tBHP treatment of ex vivo skin induces collagen-associated changes in the dermis, including fiber thinning
4. Discussion
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