R Grayson1, C W I Douglas, J Heath, A Rawlinson, G S Evans. 1. Child Health, Division of Clinical Sciences-South and Departments of Oral Pathology and Adult Health, School of Clinical Dentistry, University of Sheffield, Sheffield, UK.
Abstract
AIM: To assess the potential of gingival crevicular fluid (GCF) from adult periodontitis patients and Porphyromonas gingivalis proteases to activate matrix metalloproteinase 2 (MMP-2) in vitro. MATERIAL AND METHODS: GCF samples were collected from each of 15 adult periodontitis patients, from a clinically healthy site, a deep (>6 mm) bleeding site, and a deep nonbleeding site. The GCF samples were examined for general proteolytic activity, gelatinolytic activity and ability to activate pro-MMP-2 by zymography. Ultrasonic extracts of a range of clinical isolates of P. gingivalis cells and purified arg- and lys-gingipains were also assessed for their ability to activate pro-MMP-2. RESULTS: GCF from deep nonbleeding sites showed higher general proteolytic activity than samples from deep bleeding and healthy sites but this did not reach statistical significance. Pefabloc, a general serine protease inhibitor, inhibited the majority (92%) of the proteolytic activity. GCF samples contained neutrophil MMP-9 in its latent form in 93% of the samples, and in its activated form in 40% of the samples. In contrast, MMP-2 was present in only trace amounts in 9% of the samples. When latent MMP-2 was added to these GCF samples, it was converted to the activated form (59 kDa) in 68% of the samples. Lower molecular weight (55 and 45 kDa) activated forms also appeared in 53% of the samples, particularly those from deep sites. Activation to the 55 and 45 kDa forms was inhibited by MSAAPket (a neutrophil elastase inhibitor), whereas Pefabloc completely inhibited the activation of latent MMP-2. All ultrasonic extracts of P. gingivalis activated latent MMP-2 in a concentration- and time-dependent manner. Also, latent MMP-2 was activated by purified arg-gingipain but less efficiently by lys-gingipain. CONCLUSION: These findings suggest that P. gingivalis arg-gingipain and neutrophil elastase present in GCF can activate latent MMP-2, which may contribute in vivo to local periodontal tissue destruction.
AIM: To assess the potential of gingival crevicular fluid (GCF) from adult periodontitispatients and Porphyromonas gingivalis proteases to activate matrix metalloproteinase 2 (MMP-2) in vitro. MATERIAL AND METHODS: GCF samples were collected from each of 15 adult periodontitispatients, from a clinically healthy site, a deep (>6 mm) bleeding site, and a deep nonbleeding site. The GCF samples were examined for general proteolytic activity, gelatinolytic activity and ability to activate pro-MMP-2 by zymography. Ultrasonic extracts of a range of clinical isolates of P. gingivalis cells and purified arg- and lys-gingipains were also assessed for their ability to activate pro-MMP-2. RESULTS: GCF from deep nonbleeding sites showed higher general proteolytic activity than samples from deep bleeding and healthy sites but this did not reach statistical significance. Pefabloc, a general serine protease inhibitor, inhibited the majority (92%) of the proteolytic activity. GCF samples contained neutrophil MMP-9 in its latent form in 93% of the samples, and in its activated form in 40% of the samples. In contrast, MMP-2 was present in only trace amounts in 9% of the samples. When latent MMP-2 was added to these GCF samples, it was converted to the activated form (59 kDa) in 68% of the samples. Lower molecular weight (55 and 45 kDa) activated forms also appeared in 53% of the samples, particularly those from deep sites. Activation to the 55 and 45 kDa forms was inhibited by MSAAPket (a neutrophil elastase inhibitor), whereas Pefabloc completely inhibited the activation of latent MMP-2. All ultrasonic extracts of P. gingivalis activated latent MMP-2 in a concentration- and time-dependent manner. Also, latent MMP-2 was activated by purified arg-gingipain but less efficiently by lys-gingipain. CONCLUSION: These findings suggest that P. gingivalisarg-gingipain and neutrophil elastase present in GCF can activate latent MMP-2, which may contribute in vivo to local periodontal tissue destruction.