Matrix Metalloproteinases In Tissue Specimens Biology Essay

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Matrix Metalloproteinases family has grown to include 30 members since its first description in 1962. MMPs are zinc and calcium dependent enzymes that are capable of regulating many physiological functions by hydrolyzing the extracellular matrix under physiological pH (Banerjee et al. 2010). However, to prevent the excessive breakdown of extracellular matrix, Tissue Inhibitor metalloproteinases (TIMPs) inhibit the action of MMPs to maintain extracellular matrix homeostasis. These enzymes are involved in processes such as tissue repair, angiogenesis; in addition they take part in a number of pathological processes such as metastatic cancers, stroke, cardiovascular diseases, periodontal diseases (Lombard et al. 2005). Many years of research has been dedicated to understand the roles of different MMPs so that they can be used as therapeutic targets for drugs. However, in first clinical trials, MMP inhibitors in cancer therapy were unsuccessful. To better understand the exact role of MMPs, as they are the targets for potential drugs, different techniques for MMP assays are used to screen MMPs in tissue specimens and biological fluids. Some of the techniques discussed are immunohistochemistry (IHC), Enzyme Linked ImmunoSorbant Assay (ELISA), zymography, flow cytometry, Western Blotting (WB), (Di Girolamo, N 2010).

Immunohistochemistry (IHC) techniques are conveniently used to identify MMP production in cells of local tissues under pathophysiological conditions (Okada, Y 2001). There are two types of labeling MMPs, direct and indirect labeling. Indirect method is preferred over the direct method because it is more convenient and sensitive. In the direct method the primary antibody is applied directly to the tissue. The indirect method, the primary antibody in unlabelled and a secondary antibody will label the primary antibody. The secondary antibody is raised against the immunoglobulin of the species providing the primary antibody (Okada, Y 2001). In IHC the specific 'antigen' should be known, for example in pterygium the antigen is MMP but the MMP number should be known (Di Girolamo, N 2010). Specific recognition of MMP by primary antibodies without antibody cross-reactivity with other molecules is essential for IHC, and monoclonal antibodies are suitable for this process (PHAR 3102, 2010). In fact, monoclonal antibodies are recommended over polyclonal antibodies even though they have been used for the IHC of MMPs, however there are many different types of MMPs and therefore it is not an easy task to raise polyclonal antibodies, which are monospecific to single species of MMP (Okada, Y 2001). Before immunostaining the tissue, the fixation with formaldehyde and especially glutaraldehyde sometimes cross-links proteins so strongly that antigenic site becomes obscure and immunoreaction cannot take place. So a compromise has to be reached between the availability of the antigen and the preservation of the tissue. Monoclonal antibodies can be used for IHC in immunostaining tissues fixed with paraformaldehyde fixative. However, with formaldehyde fixed tissue, each antibody should be checked. Human tissues have to be preserved properly, so it should be handled carefully and processed at the latest within 1-3 h after surgery (Okada, Y 2001). To detect very small amounts of MMP antigens the most sensitive way to detect them in human pathological tissues is to immunostain by the Avidin-Biotin-Peroxidase Complex (ABC) and Immunogold-Silver Staining (IGSS) paraffin sections of the tissue.

ELISA kits mainly for human MMPs are widely available. The advantages of ELISA are that low sample amount is required and it can also be used to measure the amount of MMP protein, so it is a good quantitative method. However, this method cannot be used in measuring the MMP activity. Furthermore, MMP complexes/dimers are believed to play a role in MMP-associated diseases and perhaps can have an implication in the future rational drug design and the ELISA method is unable to detect the MMP dimers (Kupai, K et al. 2010). Another problem is that the antibodies, which coat the 96-microtitre plates used in ELISA, can cross-react with the sample types from other animal species. Also for ELISA soluble proteins are required, so the cells, which make different types of MMPs for example fibroblasts, macrophages, neutrophils, synovial cells, and some epithelial cells will not be identified (Kumar, V 2007).

A widely used analytical technique is western blotting (WB). WB is a semi-quantitative method used for the detection of specific proteins such as MMPs in a sample of tissue homogenate or extract (Towbin, H 1979). The steps of WB are shown in figure 1. One advantage of WB is that only a small amount of other reagents are needed to add to the protein sample extracted when loading the sample into the gel, hence this is an efficient technique. Compared to zymography procedures, the membranes used in WB are easy to handle, but similar to zymography is that both the active form and the proenzyme of MMPs can be detected by distinguishing on the basis of molecular weight (Kupai, K 2010). 'WB is a good tool to detect and characterize a many of proteins, especially those proteins that are of low abundance. The proteins immobilized on the membrane are readily and equally accessible to different ligands and multiple replicas of a gel is possible. Lastly, prolonged storage of transferred patterns, prior to use, becomes possible and the same protein transfer can be used for multiple successive analyses' (Kurien, BT & Scofield, RH 2006). The disadvantages of WB are that it is technically demanding and expensive procedure. The results are subject to interpretation, for example the presence or absence of bands and the intensity of those bands (Babu, VR 2010). Also another disadvantage is that the total amount of the detected MMP of interest in the membrane can be impeded as a result of the competition between the MMP of interest with other MMPs of a similar size within the sample (Mollica, JP 2009).

Substrate zymography is also widely used to detect MMPs. Zymography involves the electrophoresis of enzymes through polyacramide gel. Electrophoresis is performed most commonly using gelatin as the substrate copolymerized with the polyacrylamide gel (Hawkes, SP 2001). Gelatin detects MMP-2 and MMP-9 and the limit of detection of MMP-2 in gelatin zymography is very small, 10ρg hence it is very sensitive (Kupai, K 2010). The protein samples are under denaturing condition (SDS) without boiling or reducing agent. After electrophoresis, the SDS is removed from the gel or (zymogram) and is replaced by a non-ionic detergent; such as Triton X-100 this helps to renature (unfold) the proteins (Hawkes, SP 2001). The gel is incubated in a special solution for the particular proteinases under study. The zymogram is subsequently stained with Coomassie Blue and proteolytic activities are detected as clear bands against a blue background where the enzyme degraded the substrate. A particular advantage of this system is that both the active form and the proenzyme of MMPs can be detected by distinguishing on the basis of molecular weight. Other substrate zymographies follow the same protocol; however a different specific substrate is used for the MMP isoenzymes for example collagen zymography is used to detect MMP-1 and MMP-13 and casein zymography it is used to detect MMP-1, MMP-7, MMP-12 and MMP-13 (Kupai, K 2010). Recently Yu and Woessner (2001) developed a heparin-enhanced zymography technique for detection of certain MMPs, which are difficult to detect at low levels by conventional zymographies such as gelatin zymography. They are able to detect matrilysin (MMP-7) and collagenases (MMP-1 and MMP-13). A disadvantage of zymographies is that the protocol takes 2 days to follow which is quite long (Kupai, K 2010). However, the protocol is easy to follow and inexpensive. Another disadvantage of this method is that the results should be confirmed by another protein method such as WB. Also the results are semi-quantitative and not entirely quantitative and this method cannot be used to identify cells producing the MMP unless using cells in culture media, or spinal fluid, serum, synovial fluid or urine or even aqueous humour, (Di Girolamo, N 2010). Zymography is used to detect MMPs in biological fluids, but similar to ELISA the cells that MMP activity is coming from are unknown.

Finally another used method for both clinical and research purposes of screening MMPs, is flow cytometry. This procedure involves, the analysis of biological fluids containing cells such as synovial fluid. The primary system of the flow cytometry has two major components, the fluidic system which presents particles or cells one at a time to the interrogation point and takes away the wastes (Introduction to flow cytometry 2010). The laser is the light source for scatter and fluorescence, and the amount of forward and sideward scatter directly indicates the size and internal complexity of the cells (Pedreira, CE et al. 2008). Flourocrome-conjucated monoclonal antibodies recognize the MMPs expressed in specific populations of the cell. The expression of several MMPs for each cell can be analyzed with the currently available clinical flow cytometers (Pedreira, CE et al. 2008). The primary advantage of flow cytometry is that multiple parameters can be evaluated in millions of cells in a few seconds and the information about the cell can be stored digitally. Also, like ELISA and IHC small amount of the sample is required. The major disadvantage of this system is that it is very expensive to run and the preparation of the samples requires reagents, which are expensive as well (Curren, AD 1999).

There is no doubt that the above mentioned techniques have been, and continue to be valuable experimental tools for the detection of MMPs, and subsequently, the discovery of knowledge in various fields of research. There are advantages and, ofcourse, disadvantages in all of the techniques discussed above, and those that have not been discussed such as ELISA. Until more improvements are made to these techniques, therefore, researchers should choose the method according to their interests, as well as perform the techniques with precision and care.

There is no doubt that the above mentioned techniques have been, and continue to be valuable experimental tools for the detection of MMPs, and subsequently, the discovery of knowledge in various fields of research. There are advantages and, ofcourse, disadvantages in all of the techniques discussed above, and those that have not been discussed such as ELISA. Until more improvements are made to these techniques, therefore, researchers should choose the method according to their interests, as well as perform the techniques with precision and care.

Western Blotting technique

Taken from: Thomas, S 2010

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