In Situ Zymography of Gelatinase Activity
During the past decade, in situ zymography has been applied to localize gelatinase activity in tissue sections (Galis et al. 1994). This method is based on the principle of gel substrate zymography. Galis et al. (1994)(1995) used gelatin that contains fluorescein or an autoradiographic emulsion as substrate layer on cryostat sections of human atherosclerotic plaques to assess gelatinolytic activity in situ. Autoradiographic emulsions were introduced because gelatin is the main component of the emulsion and gelatinolytic activity during incubation results in decreased amounts of silver in specific sites that can be visualized by photographic development. Microscopy reveals transparent spots on top of the areas with gelatinolytic activity against a black background (Galis et al. 1994). Fluorescein-coupled gelatin was introduced to try to improve the precision of localization of gelatinolytic activity because disappearance of fluorescence indicates areas with gelatinolytic activity (Galis et al. 1995). Because fluorescein is a fluorophore that does not have optimal fluorescent properties for microscopy, Oregon Green conjugates were introduced, which have similar spectral characteristics as fluorescein, but their fluorescence is more photostable and less pH-dependent than fluorescein (Pirila et al. 2001; Faia et al. 2002).
The principle introduced by Galis et al. (1995) has been modified in the procedure to demonstrate breakdown of gelatin differently. Instead of fluorescent gelatin, some authors used pure gelatin that was stained after incubation with Ponceau S (Loy et al. 2002), amido black (Ikeda et al. 2000; Furuya et al. 2001), or Biebrich Scarlet (Wada et al. 2003). Another approach based on zymography was used by Kurschat et al. (2002), who incorporated gelatin into polyacrylamide gels of 50 µm thickness, which were brought into contact with unfixed cryostat sections. After incubation, sections and gels were separated and gels were stained with Coomassie Blue. All the approaches have in common the fact that a decrease in staining intensity is a reflection of gelatinolytic activity. Details of the principles of photographic emulsion-based and fluorescently-labeled substrate-based in situ zymography were recently reviewed by Yan and Blomme (2003). These authors concluded that the photographic emulsion technique was more sensitive than the fluorescent gelatin principle.
The approach of gelatin in situ zymography has been applied to study involvement of gelatinolytic activity in many (patho)physiological processes in tissues such as arteries (Knox et al. 1997; Bruno et al. 1998; Faia et al. 2002), veins (Fernandez et al. 1998; George et al. 1998; Kranzhofer et al. 1999), heart (Tyagi et al. 1996; Robert et al. 1997), lung (Pardo et al. 1996; Leco et al. 2001), skin (Fisher et al. 1997; Krejci-Papa and Paus 1998), eye (Zhou et al. 1998; Hanyu 1999), equine hoof (Mungall et al. 1998; Mungall and Pollitt 1999,2001), joints (Freemont et al. 1999; Yamanaka et al. 2000), colon (Tarlton et al. 2000), muscle (Kieseier et al. 2001), nerve (Duchossoy et al. 2001; Siebert et al. 2001), ovary (Curry et al. 2001; Khandoker et al. 2001), gingiva (Pirila et al. 2001), adipocytes (Maquoi et al. 2002), and endometrium (Zhang and Salamonsen 2002; Zheng et al. 2002). All studies indicate that gelatinases have a role in remodeling and/or degradation of the ECM.
Based on the high-level expression and proenzyme activation of gelatinases in tumors, in situ zymography has been used to study the involvement of gelatinolytic activity in cancer progression. Gelatinolytic activity was demonstrated in a series of human malignancies such as those of ovary (Furuya et al. 2001; Lengyel et al. 2001), cervix (Minami et al. 2003), breast (Iwata et al. 2001), lung (Ikeda et al. 2000; Kaji et al. 2003), thyroid (Nakamura et al. 1999), esophagus (Koyama et al. 2000), oral cavity (Shimada et al. 2000), brain (Nakada et al. 1999), kidney (Kamiya et al. 2003), and liver (Kaneyoshi et al. 2001). In almost all cases, MMP-2 appeared to be responsible for gelatinolytic activity and the activity was presumed to be related to the invasive and metastatic properties of the cancers.
The approach that was used in these studies was reduction in staining intensity of the gels on top of the sections. This approach has two major disadvantages. First, sensitivity of reduction in staining intensity is less than that of formation of staining and, second, it is doubtful whether this principle can be used for quantitative purposes (Thomas et al. 1998; Mungall and Pollitt 2001). Therefore, other procedures were developed in which a colored or fluorescent product was formed at the site of gelatinolytic activity. Ratnikov et al. (2000) introduced biotinylated gelatin as a substrate to demonstrate gelatinolytic activity in solution. After cleavage of the substrate, the proteolytic fragments bearing the biotin moieties are captured by streptavidin coated on the plastic surface of a 96-well microtiter plate. Activity of horseradish peroxidase conjugated to streptavidin is measured. This biotinylated gelatin may also have perspectives for in situ localization when diaminobenzidine or aminoethylcarbazole is used to localize peroxidase activity.
The application of gelatin as substrate for in situ zymography has the advantage that, as far as we know, only MMP-2 and MMP-9 have a high affinity for this substrate. However, definite conclusions about the specific enzyme(s) responsible for gelatin breakdown can be drawn only when selective inhibitors are used and the in situ zymography is combined with gelatin zymography and IHC of MMP-2, MMP-9, and other potential gelatin-degrading enzymes. Gelatin zymography enables the assessment of molecular weights of the proteins that degrade gelatin. Moreover, differences in molecular weight of proenzymes and activated enzymes allow estimation of relative amounts of proenzymes and active enzymes in homogenates of tissues under study.
It can be concluded that in situ zymography with gelatin as substrate enables the localization of MMP activities, but thus far precise localization is not possible.