The protein expression network in periodontal pocket tissue: a preliminary study

Background and Objective The periodontal disease (PD) is caused by a set of inflammatory disorders characterized by periodontal pocket formation that lead to tooth loss if untreated. Periodontitis diagnosis is only based on clinical assessment only, in the absence of a reliable pathogenic check based on appropriate interpretation of inflammation. A modern pathogenic model based on a multilevel framework including disease-initiating and -resolving mechanisms is requested. Studies on PD utilizing proteomic analysis have been performed on saliva or crevicular fluid samples, peripheral blood or periodontal plaque samples, but not on the pathologic tissue of the periodontal pocket, which is the key lesion of the PD. The aim of this work was to compare the proteomic profile of the pathologic interproximal gingival pocket tissue with the corresponding of interproximal gingival healthy tissue, obtained from sites where no periodontal-pathogenic bacteria were detectable. Materials and methods Twenty healthy subjects (T, test group), affected by chronic PD and twenty periodontally healthy subjects (C, control group), were enrolled in the study. T subjects underwent to the periodontal resective treatment, while C subjects underwent to the crown lengthening surgical treatment. To characterize the proteomic profile of periodontally-affected patients, their interproximal periodontal pocket tissue was compared with that of periodontally-healthy patients. Pocket-associated and healthy tissue samples, harvested during surgical therapy, were treated to extract the protein content. Tissues were always collected at sites where no periodontal-pathogenic bacteria were detectable. 2DE (Two-dimensional gel electrophoresis) and LC-MS/MS (Liquid Chromatography-tandem mass spectrometry) analysis were performed for T and C gingival tissue samples to separate and identify the proteins. Web-based bioinformatics tools (iPROClass and CateGOrize) were employed to investigate all potential localizations, molecular functions and biological processes of the identified proteins. After identification, proteins were selected for subsequent Western Blot quantitation both in pathological and healthy tissues. Results The identified proteins are mainly involved in metabolism (32 %), transport (13 %) and cell organization and biogenesis (13 %). A significant unbalance in protein expression between healthy and pathological sites was recorded. Thirty-two protein spots were overall identified, and four proteins, the protein S100-A9 (S100A9), heat shock protein beta-1 (HSPB1), Galectin-7 (LEG7) and 14-3-3 protein zeta/delta (14-3-3) were selected for Western blot analysis of both periodontally-affected and healthy patients. The four selected proteins resulted extremely over-expressed in periodontal pocket tissue when compared with the corresponding tissue of periodontally-healthy patients. Discussion: S100A9 is involved in the regulation of inflammatory processes, immune response, oxidant-scavenging and apoptosis-inducing activities. HSPB1 synthesis increases in response to a variety of stresses (e.g. elevated temperatures, heavy metals, toxins, oxidants, bacterial and viral infections) in order to minimize the attendant deleterious consequences. LEG7 expression contributes to the tissue remodeling processes following tissue damage and protects cell from death. The 14-3-3 proteins are involved in the control of several cell cycle checkpoint, connective tissue remodeling, apoptosis signaling, during inflammation response. The Western blot and proteomic analyses are congruent with the reaction of injured periodontal tissues in PD. The proteomic analysis was performed for the first time directly on periodontal pocket tissue. The proteomic network highlighted enhances the understanding of PD pathogenesis revealing a defensive protein strategy which, however, evidently fails in patients affected by PD. Further study are requested also for specific therapeutic strategies setting.