Resumen de High Concentration but Low Activity of Hepatocyte Growth Factor in Periodontitis
Background: High levels of hepatocyte growth factor (HGF), a healing factor with regenerative and cytoprotective effects, are associated with inflammatory diseases, including periodontitis. HGF biologic activity requires binding to its receptors, the proto-oncogene c-Met and heparan sulfate proteoglycan (HSPG). This study investigates HGF expression and its relationship to subgingival microbiota in medically healthy individuals with and without periodontitis.
Methods: Saliva, gingival crevicular fluid (GCF), and blood samples from 30 patients with severe periodontitis and 30 healthy controls were analyzed for HGF concentration using enzyme-linked immunosorbent assay and binding affinity for HSPG and c-Met using surface plasmon resonance. The regenerative effects of saliva from three patients and controls were analyzed in an in vitro model of cell injury. Subgingival plaques were analyzed for the presence of 18 bacterial species.
Results: Patients with periodontitis showed higher HGF concentrations in saliva, GCF, and serum (P <0.001); however, the binding affinities for HSPG and c-Met were reduced in GCF and saliva (P <0.002). In contrast to the controls, saliva from patients showed no significant regenerative effect over time on gingival epithelial cells. Compared with controls, patients had a higher prevalence of periodontally related bacteria.
Conclusions: Higher circulatory HGF levels indicate a systemic effect of periodontitis. However, the HGF biologic activity at local inflammation sites was reduced, and this effect was associated with the amount of periodontal bacteria. Loss of function of healing factors may be an important mechanism in degenerative processes in periodontally susceptible individuals.
Periodontitis is the pathologic manifestation of host responses against bacterial challenges from the dental biofilm at the tooth-gingiva interface.1 It is characterized by gingival inflammation, tissue breakdown, connective tissue attachment loss, and pocket formation, ultimately leading to tooth loss. Gingival inflammation leads to continuous release of a cascade of biologically active substances. In patients with periodontitis, specific periodontal microbiota are associated with higher levels of inflammatory biomarkers in gingival crevicular fluid (GCF) and saliva.2 Furthermore, elevated levels of circulatory inflammatory mediators3 and the presence of periodontal bacteria at systemic locations4 link periodontitis to systemic diseases with inflammatory backgrounds, such as cardiovascular disease, kidney disease, and rheumatoid arthritis.5-7 Chronically inflamed periodontal pockets may serve as inflammatory stimulus reservoirs from which oral bacteria and their components can enter the circulation, activating neutrophils and platelets8 and triggering the inflammatory process in the vessels.
Hepatocyte growth factor (HGF) is a potent multifunctional healing factor involved in the repair and regeneration of various tissues and organs, such as in coronary artery disease (CAD), kidney disease, and skin ulcers.9-11 HGF concentrations in GCF and saliva increase proportionally with periodontitis progression,12 and HGF is considered to be a biomarker of periodontal disease severity.13 HGF production in human gingival fibroblasts is stimulated by proinflammatory cytokines, such as interleukin (IL)-1, and by bacterial virulence factors, including gingipains and fimbriae of Porphyromonas gingivalis and lipoteichoic acid from Streptococcus sanguinis.14-16 In response to P. gingivalis, human gingival fibroblasts in vitro also produce other inflammatory cytokines, such as IL-6, IL-8, and tumor necrosis factor (TNF)-a.17 In contrast, it has been shown that P. gingivalis inhibits IL-8 and IL-6 expressions,18 partially due to proteolytic activity.
The HGF receptor is the proto-oncogene c-Met, a transmembrane tyrosine kinase receptor expressed by almost all epithelial and endothelial cells (mesenchymal cells).19 Heparan sulfate proteoglycan (HSPG), a glycosaminoglycan present on the cell surface in essentially all tissues and in the extracellular matrix,20 includes a low-affinity binding site that functions as a co-receptor for c-Met/HGF.21 Binding to HSPG is essential for the interaction of HGF with c-Met and induction of cellular responses. It also facilitates cytokine/c-Met receptor interaction,22 the conversion of promitogen HGF to the two-chain mature form,23 and excretion of excess HGF from blood.24 A number of studies have demonstrated elevated HGF in patients with various chronic diseases.25,26 However, this overexpressed HGF may exist in a form with reduced biologic activity, characterized by reduced binding affinity to HSPG or the glycosaminoglycan dextran sulfate (DS), which is associated with decreased regeneration in a model of cell injury of mouse skin epithelial cells (CCL-53.1).27,28 Neutrophils release proteases that reportedly cleave and inactivate HGF, leading to inhibited c-Met signaling in patients with chronic non-healing wounds29 and reducing HGF binding to HSPG during inflammation.30 P. gingivalis also decreases the binding affinity of HGF to HSPG.10 In a previous study of patients with CAD, the present authors found a tendency for HGF to exhibit slightly reduced biologic activity in individuals with periodontitis and prevalent P. gingivalis, compared with patients who had CAD but not periodontitis or P. gingivalis.31 HGF is elevated in association with c-Met receptor expression,32 suggesting that it may be involved in periodontitis pathogenesis and progression. In the environment of periodontitis, connective tissue attachment loss impairs proper regeneration of deep collagenous structures in the periodontium, and HGF may work as a chemotactic and proliferative factor for gingival epithelial cells.
The enzyme-linked immunosorbent assay (ELISA) is a reliable method for protein detection and concentration measurement; however, it cannot differentiate between biologically active and inactive HGF.27 Surface plasmon resonance (SPR) is an optical technique that can determine the affinity of a protein for several ligands or epitopes.33 SPR-based assessment of the binding profile of HGF to HSPG and other ligands can rapidly and sensitively differentiate HGF variants with different biologic activities and is an appropriate method for evaluating the quality of endogenous HGF in clinical studies.28 The present study aims to investigate the concentration and biologic activity of HGF in patients with severe periodontitis and examine the relationship between HGF expression and subgingival microbiota.
