Expression of markers of type 2 diabetes-induced premature cellular senescence in gingival biopsies of individuals with periodontal health and periodontitis

University Of Florida, Gainesville FL

Investigators

Abstract

Background. Cellular senescence is a process in which cells become irreversibly cell cycle arrested and resistant to apoptosis, but still metabolically active with a pro-inflammatory secretome, named senescence- associated secretory phenotype (SASP). Diabetes (DM) complications have been related to premature age- independent accumulation of senescent cells in tissues. Previous evidence suggests that harmful effects of DM on periodontal tissues are partly due to a hyper-inflammatory phenotype, though the full mechanisms behind this process remain undefined. Thus, further investigation into the cellular and molecular mechanisms linking DM to periodontal disease pathogenesis is needed. Our main hypothesis is that healthy and diseased periodontal tissues in subjects with type 2 DM (T2DM) present altered expression of markers of senescence, when compared to those of normoglycemic individuals. Hence, T2DM-induced premature senescence may represent a mechanism by which the periodontal tissues of subjects with T2DM is injured. This proposal will use two specific aims to address this hypothesis. Aim 1. To compare the expression of cellular senescence markers in healthy and diseased gingival biopsies between young/middle-aged individuals with T2DM and those who are normoglycemic, and to investigate whether the combination of T2DM and periodontitis impacts cellular senescence markers beyond their individual effects. Gingival tissues will be collected from young/middle-aged subjects according to the following groups: 1- without periodontitis or DM, 2- without periodontitis but with T2DM, 3- with periodontitis but without DM, and 4- with both periodontitis and T2DM. SA-β-Gal and lipofuscin staining will be used to grossly quantify the amount of senescence within the tissues. In order to quantify the expression of makers of senescence within the tissues, Luminex® technology and QuantiGene Plex assays will assess the mRNA levels of p16INK4a, p14ARF, p53, p21, Rb, HMGB1, H2AX, P53BP1, LMNB1, HP1α, ATR, ATM, Chk1, Chk2, BCL-2, BCL-XL, and HIF-1α. Moreover, immunohistochemistry and immunofluorescence will be used to probe for protein levels of p16INK4a, p14ARF, p53, p21, H2A.X, P53BP1, laminin B1, HP1α, ATR, ATM, BCL- 2, and HIF-1α. In order to characterize and quantify the mRNA and protein levels of SASP factors within the biopsies, two-pronged Luminex® technology approaches will be taken, the QuantiGene Plex and ProcartaPlex assays. Aim 2. To correlate markers of senescence with the local levels of inflammatory mediators. This analysis will be coupled with that performed in Aim 1 to take a systems biology approach to correlate markers of senescence with the levels of various immunoinflammatory mediators by the Luminex® technology and 80-Plex ProcartaPlex Panel. Latent Class Analysis (LCA) will identify phenotypes of senescence within each experimental group and describe the features of each phenotype identified. Within each LCA defined phenotype, GO analysis will be performed using DAVID database to determine whether T2DM, periodontitis and inflammatory microenvironment affects genes or proteins associated with senescence.