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  • Periodontitis: evaluation of enzyme activity tests MMP-8

Matrix metalloproteinases (MMPs)

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, produced by many cell types including fibroblasts, endothelial cells, osteoblasts, macrophages, lymphocytes and neutrophils. MMPs are able to degrade several extracellular matrix components (ECM), but also cytokines, receptors and the factors that regulate cell motility.

MMPs are the main proteolytic enzymes involved in the remodeling and degradation of extracellular matrix components, in the modifications of interactions between cells, and of those between cells and the ECM that regulate such processes such as cell migration. For these features, MMPs are involved in many physiological (angiogenesis, apoptosis, bone remodeling, wound healing, morphogenesis, inflammation, immune response, etc.) and pathological processes (periodontitis, arthritis, cancer, cardiovascular and neurological disease, osteoporosis etc.).

Today there are at least 26 known human metalloproteinase which on the basis of their structure, of the substrate specificity and of the cellular localization, are classified into: collagenase (MMP-1, -8, -13 and -18), gelatinases A and B (MMP -2 and -9), stromelysin, matrilysin and membrane metalloproteinases (MT-MMP).


Structure

Generally the structure of MMPs contains a N-terminal signal peptide necessary for proper secretion, a pro-domain which touches the zinc ion of the catalytic site to keep the MMP in an inactive form, a catalytic domain that includes the binding sites with zinc, and a hinge region followed by an hemopexin type C-terminal domain.

MMP-2 and MMP-9 gelatinases possess an additional fibronectin type II domain, inserted into the catalytic domain that is involved in the binding of substrates like for example, collagen and gelatine.

MT-MMPs contain an additional C-terminal transmembrane domain that anchor them to the cell surface.


Regulation of MMPs expression and activation

Constitutive expression of the MMPs encoding genes is low. However, in all those physiological or pathological conditions that require a remodeling of the ECM, an increased synthesis regulated at transcriptional and post-transcriptional level is induced. Among the factors which influence the transcription of MMPs genes, there are pro-inflammatory cytokines, growth factors, hormones, oncogenes and tumor promoters. In vitro the expression of several MMPs can be induced by IL-1, IL-6, TNF-α, EGF, PDGF, FGF e TGF-β. Depending on the cell type involved, other cytokines, such as IL-4, IL-10, INF-γ e lo stesso TGF-β, possono, a seconda del tipo cellulare coinvolto, may decrease the expression of MMPs.

Most of the MMPs are released from cells in a latent form as pro-enzyme (pro-MMP). The conversion of pro-MMPs in active form requires a specific activation process called "cystein switch". In pro-domain there is an unpaired cysteine residue that binds to the zinc ion located in the active site. The cleavage of the pro-domain results in the destruction of the bond zinc-cysteine and the loss of N-terminal pro-domain, turning the active site accessible. For most of the MMPs proteolytic activation starts in the extracellular space with serine proteases such as plasmin, plasminogen activator of the urokinase type (uPA) or other members of the family of MMPs. On the cell surface also the MT-MMPs have been identified as strong physiological activators of some MMPs.

The MMPs activity is also regulated by the family of tissue inhibitors of MMPs (TIMP), which includes at least four members (TIMP-1/TIMP-4). TIMPs found in tissues or fluids such as serum and saliva, are able to bind to the active site of both pro-MMPs and active MMPs, inhibiting respectively the autocatalytic activation and the proteolytic activity.

A complex system of regulation protects the conservation of the balance between expression and inhibition of MMPs ensuring proper remodeling of the extracellular matrix that is essential in the processes of tissue morphogenesis, repair and apoptosis. A poorly regulated enzyme activity that supports the process of degradation of the matrix instead, will result in a fibrogenic response that is no longer able to compensate for the proteolytic activity.

It has been shown that an imbalance between MMPs and TIMPs causes degradation of cartilage observed in arthritis and osteoarthritis as well as promoting bone resorption. At periodontal tissue level TIMPs can be synthesized by several cell types such as fibroblasts, osteoblasts, endothelial cells, monocytes, macrophages and keratinocytes. Also the destruction of periodontal tissue has been correlated with an increased proteolytic activity at periodontal level when not properly controlled by inhibitors.

MMPs can be therefore considered as promising therapeutic targets and evaluating their activities is essential in monitoring many pathological conditions.


Metalloproteinase-8

Collagen is one of the major components of connective tissue and extracellular periodontal matrix. The correct and constant renewal of the components in the extracellular matrix can make the difference between health and periodontal disease in which is noted an imbalance between synthesis and degradation. In the course of gingivitis collagen fibers are degraded promoting the infiltration of inflammatory cells to the source of infection, resulting in gingival redness and swelling. The chronic inflammatory state promotes the onset of periodontal disease symptoms with further destruction of the collagen fibers at periodontal ligament level and loss of bone tissue.

The process of collagen degradation is largely performed by MMPs.

MMP-8 is also known as neutrophil collagenase as it was considered to be exclusively produced by this cell type. Actually scientific research has shown that MMP-8 is also produced by endothelial cells, smooth muscle cells, macrophages and more specialized cells such as the odontoblasts, the gingival fibroblasts, periodontal ligament cells and gingival sulcus. It has been proven that synthesis and release of these cells requires prolonged exposure to pro-inflammatory cytokines (IL-1, TNF-α, IL-8) or bacterial LPS, while MMP-8 produced as pro-enzyme and stored in specific granules from neutrophils, is released instantly in the extracellular space after a pro-inflammatory stimulation, where it is activated by the removal of the pro-domain.

The main target of MMP-8 is type I collagen of tendons, bones, dentin and cement; type II of cartilage; type III that constitutes skin, muscles, blood vessels.


MMP-8 and periodontal disease

Periodontal disease and peri-implantitis are characterized by the progressive destruction of teeth supporting tissues and implants, resulting from the inflammatory response due to bacterial infection. MMP-8 is mainly synthesized by neutrophils, the most important immune system cells for defense against periodontal bacteria. For its enzymatic properties the AMMP-8 degrades the type I, II and III collagen fibers promoting the destruction of periodontal tissues. Several studies emphasize the presence of high levels of metalloproteinases, especially the MMP-8, in the crevicular fluid, saliva or in biopsy specimens of periodontal patients. Conversely, in non-periodontal patients or patients with gingivitis significant levels of AMMP-8 are not found.

The presence in the oral cavity of AMMP-8, correlates with the progression and with the severity of periodontal clinical parameters: gingival bleeding, loss of attachment and deeper space of the pockets. Studies in literature also show that the assessment of the concentration of salivary AMMP-8 allows the identification of periodontal patients with a significant loss of alveolar bone. In addition, a reduction in the concentration of salivary AMMP-8 is connected to a successful periodontal therapy and to the improvement of clinical parameters. The elimination of bacterial infection responsible for periodontal and peri-implant disease treats the inflammatory process resulting in a negative feedback on the activation of MMP-8 that therefore, as a result of an effective therapeutic treatment, is again not detectable in salivary level.

The activation of MMP-8 anticipates the tissue damage and its recognition in the crevicular fluid or in the peri-implant sulcus is an important biomarker for early diagnosis of periodontal and peri-implant disease, as well as a clear indicator of therapeutic efficacy.

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