Uncoupled inflammatory, proliferative, and cytoskeletal responses in senescent human gingival fibroblasts

Abstract

Background and objective: Aging is characterized by a decline in tissue structure and function that may be explained by the development of cellular senescence. However, the acquisition of specific phenotypic responses in senescent gingival fibroblasts is still poorly understood. Here, we have analyzed whether proliferation of primary cultures of human gingival fibroblasts may affect different cell functions relevant to cellular senescence and tissue deterioration.

Methods: Human gingival fibroblasts from five young donors were expanded until cellular senescence was achieved. Cellular senescence was evaluated by determining modifications in cell size, cell proliferation, p16 and p21 mRNA levels, H2Ax phosphorylation, cell viability, and senescence-associated beta-galactosidase staining. Inflammation was evaluated by analyzing the secretion of cytokines and nuclear translocation of NF-kappa B. Collagen remodeling was evaluated using a collagen gel contraction assay. Immunofluorescence and confocal microscopy were used to determine changes in the localization of the cytoskeletal proteins. Data analysis was performed to identify changes between cultures of the same donor at early and late passages using the paired sample t test or the Wilcoxon matched-pairs signed-rank test.

Results: Late passage cells showed changes compatible with cellular senescence that included increased cell size, reduced cell proliferation, staining for SA-beta gal, phosphorylated H2Ax, and increased p16 and p21 mRNA levels. Late passage cells showed a decrease in collagen contraction and reduced co-localization between the cytoskeletal proteins actin and vinculin. Importantly, late passage cells neither demonstrated changes in the secretion of inflammatory cytokines nor NF-kappa B activation.

Conclusion: Our results imply that cytoskeletal changes and inhibition of cell proliferation represent early modifications in the structure and function of senescent gingival fibroblasts that are not coupled with the acquisition of an inflammatory phenotype. Further studies are needed to clarify the impact of different senescence stages during aging of the periodontium.