Inflammatory colorectal polyps in dogs are characterized by the presence of multiple polyps in the colorectal area with dense infiltration of inflammatory cells, such as neutrophils and macrophages.1 This disease is common in MDs in Japan.1 Previous studies have indicated that dogs with ICRPs respond to anti-inflammatory and immunosuppressive treatments with drugs such as prednisolone, cyclosporine, and lefunomide.1,2 Molecular analyses have revealed that ICRP lesions in MDs have inflammatory cytokine profiles similar to those observed in inflamed intestinal tissue samples obtained from humans with IBD, indicating that ICRPs of MDs represent a type of IBD.3
In the disease process of IBD, proteases have an important role in degradation of the ECM, promotion of cell migration, facilitation of tissue remodeling, and regulation of cell function by cleaving cytokines, chemokines, receptors, other proteases, and adhesion molecules.4–6 Metalloproteinases are a family of endopeptidases containing Zn2+ that are classically assigned to one of several categories, including collagenases, gelatinases, stromelysins, and matrilysins, according to their substrate.7 Metalloproteinase-2 and MMP-9, which are gelatinases, degrade gelatin, elastin, fibronectin, various types of collagens (types I, IV, V, VII, X, and XI), laminin, aggrecan, and vitronectin8 and have been shown to have increased expression in IBD-related lesions in humans.9,10 Metalloproteinases are secreted in a latent form (pro–MMP) and activated by a multistep process in which a protease-susceptible region of the pro–MMP is first cleaved by several proteases.6,11,12 These proteases include serine proteases, which are fundamental mediators of intestinal homeostasis under physiologic conditions. In pathophysiologic situations, such as cases of IBD, dysregulation of proteolytic homeostasis can occur; increases in serine protease activities in IBD-related lesions in humans have been reported.13
Although MMP-2 and MMP-9 activities are increased in the intestinal mucosa of dogs with chronic enteropathy, compared with their activities in the intestinal mucosa of healthy Beagles,14 there is no report of MMP activities in the intestinal mucosa of dogs with ICRPs. Moreover, serine protease activities in the intestinal mucosa of dogs have not been studied to our knowledge. Therefore, the purpose of the study reported here was to investigate the activities of MMP-2, MMP-9, latent forms of MMP-2 and MMP-9 (pro–MMP-2 and pro–MMP-9), and serine proteases in samples of inflamed colorectal mucosa of MDs with ICRPs. Our hypothesis was that MMP-2 and MMP-9 activities as well as serine protease activities would be increased in the ICRP-related colorectal mucosal lesions of MDs, compared with findings for samples of unaffected colorectal mucosa from MDs with ICRPs and samples of colorectal mucosa from dogs with non–ICRP-related large bowel diarrhea.
Materials and Methods
Animals
Fifteen MDs with ICRPs were included in the study. The diagnosis of ICRPs was obtained on the basis of histopathologic findings, according to a method used in previous reports.1,15 Five dogs that were referred to our hospital for investigation of the cause of large bowel diarrhea and were found to have non– ICRP-related disease on the basis of histopathologic findings were considered as controls. A signed consent form was obtained from the owner of each dog for tissue sample collection and usage in the study. All procedures on dogs were approved by the Hokkaido University Institutional Animal Care and Use Committee.
Tissue sample collection
For sample collection, endoscopic examinations were performed after the dogs were anesthetized. For each dog, midazolam (0.1 mg/kg) and butorphanol tartrate (0.2 mg/kg) were administered IV as pre-medication, followed by IV administration of propofol (4 to 6 mg/kg). Then, anesthesia was maintained via inhalation of isoflurane with oxygen. Additional doses of butorphanol were administered during the endoscopic examinations, if necessary. For the MDs with ICRPs, fresh colorectal mucosa samples that appeared inflamed were collected endoscopically. In addition, noninflamed colorectal mucosa tissue samples that appeared macroscopically normal were collected endoscopically from the MDs with ICRPs. The noninflamed tissue samples from MDs with ICRPs were collected from a region of colorectal mucosa adjacent to grossly normal mucosal tissue, according to the histopathologic standards established by the World Small Animal Veterinary Association Gastrointestinal Standardization Group.16 Colorectal mucosa tissue samples were similarly collected endoscopically from the control dogs. At least 6 tissue samples each were collected from inflamed and noninflamed colorectal mucosal regions of dogs with ICRPs and from noninflamed colorectal mucosal regions of control dogs. One of each type of tissue from each dog was stored at −80°C and used for zymography, and the remaining samples were used for histologic and immunohistochemical analyses. Electrocardiography, pulse oximetry, and capnography and assessments of arterial blood pressure and rectal temperature were performed to monitor each dog's condition during the procedure. Endoscopic procedures were finished within 2 hours for all dogs. No dogs developed any complications after the sample collection procedure, and no postprocedural treatments were required.
Gelatin zymography
Samples for gelatin zymography were prepared as described previously17 with some modifications. Briefly, a snap-frozen colorectal sample of each type from each dog (inflamed and noninflamed colorectal mucosa from dogs with ICRPs and noninflamed colorectal mucosa from control dogs) was homogenized individually in a buffer solution (20 μL/mg tissue) including 50mM Tris base, 150mM NaCl, 10mM CaCl2, 0.01% Triton × (pH, 7.6), and protease inhibitor cocktail tablets.a Then, the samples were centrifuged at 13,000 × g at 4°C for 10 minutes. After centrifugation, the supernatants were collected, and protein concentrations were measured with a protein assay bicinchoninate kit.b
Gelatin zymography was performed with a gelatin zymography kit,c according to the manufacturer's instructions. This kit was designed to detect the gelatinolytic activities of MMP-2 and MMP-9 as well as their latent forms (pro–MMP-2 and pro–MMP-9) in tissue samples from any animal species. Briefly, each sample (10 μg of total protein with an equal amount of the loading buffer) was loaded onto precast gels. Each sample was loaded in duplicate. The MMP markers (human pro–MMP-2, MMP-2, and pro–MMP-9) provided by the manufacturer were also loaded on each gel as standards. Then, electrophoresis was performed. After the run was completed, the gels were put into a washing buffer and incubated with shaking at room temperature (approx 25°C) for 1 hour. Then, the gels were put into the reaction buffer in a sealed container and incubated at 37°C for 20 hours. After the enzymatic reaction, the gels were placed in staining solution and incubated at room temperature for 30 minutes. Then, the gels were put into destaining solution. The gelatinase activities of the MMPs were seen as clear bands on the gels. For the analysis of MMP activities, the gels were scanned, and the intensity of each band was measured with imaging software.18,19,d The intensity of each band was quantified by densitometry, as described previously, which generated the peak area of the band.18 When the peak area had several peak levels, these peaks were analyzed separately as different activities. The intensities of the bands are reported as AU; values were calculated by comparing the intensity of the band to that of the standard band on each gel. Because the MMP markers provided by the manufacturer did not include a marker for MMP-9, the intensity of the MMP-9 band was compared with that of the standard pro–MMP-9 band. All measurements are expressed as the mean of duplicate runs of 1 sample of each type of tissue for each dog.
Serine protease zymography
For serine protease zymography, all procedures were performed in a manner similar to those used for gelatin zymography. Serine protease zymography was performed with a serine protease zymography kit,e according to the manufacturer's instructions. This kit was designed to detect the activities of various serine proteases, such as plasmin, trypsin, elastase, and cathepsin G, in tissue samples from any animal species. The same homogenized sample used for gelatin zymography was also used in serine protease zymography. As a standard, 8 ng of human plasminf was loaded onto each gel. Because various serine proteases can be detected by serine protease zymography, the activities of serine proteases were measured as total proteolysis (SNBI) per lane as described previously9 and are reported as AU; values were calculated by comparing the intensity of the total activity to the activity intensity of the plasmin standard on each gel. All measurements are expressed as the mean of duplicate runs of 1 sample of each type of tissue for each dog.
Immunohistochemical analyses
The localization of MMP-2 and MMP-9 in sections of inflamed colorectal mucosa obtained from MDs with ICRPs (n = 10 dogs) and in sections of colorectal mucosa obtained from control dogs (5 dogs) was investigated by immunohistochemical analyses. These tissue samples were fixed in neutral-buffered 10% formalin and embedded in paraffin for sectioning. After sectioning, these samples underwent a deparaffination process and were soaked in 0.01M citrate buffer (pH, 6.0) and autoclaved (at 110°C) for 15 minutes for antigen retrieval. After treatment with 0.3% hydrogen peroxide in methanol for 20 minutes to block endogenous peroxidase activity, the tissue sections were incubated with 10% nonimmune normal goat serumg for 1 hour to block nonspecific reactions. The primary antibodies used were mouse anti–MMP-2 monoclonal antibody (1:6,400)h and rabbit anti–MMP-9 monoclonal antibody (1:6,400)i; all tissue sections were reacted overnight (approx 18 hours) at 4°C. Because the mouse anti–MMP-2 monoclonal antibody used had not been shown to react with canine MMP-2, we confirmed the reactivity of this antibody in canine osteosarcoma tissue samples (data not shown); such tissue samples have a high level of MMP-2 expression.20 The tissue sections were washed and then reacted with a biotinylated anti-mouse IgG antibody or biotinylated anti-rabbit IgG antibody for 30 minutes. After incubation of the tissue sections with streptavidin for 30 minutes, 3,3′-diaminobenzidine was added to the tissue sections. Mayer hematoxylin counterstain was applied. For negative controls, unconjugated affinity-purified normal mouse or rabbit IgG antibodyj was used instead of the primary antibodies.
Immunohistochemical analyses involved assessment of tissue section staining with a previously described semiquantitative scoring system10 with some modifications. Sections were examined microscopically by one of the authors (NN). One section of each sample collected from each dog was stained with each primary antibody and examined. Each section was assigned a staining score as follows: 0 (no staining), 1 (staining of few cells or areas or a very weak staining in all cells), 2 (staining of a minority of cells or areas or a weak staining in all cells), 3 (staining of most cells or areas or moderate staining in all cells), and 4 (strong staining of all cells or areas). For semiquantification, 3 randomly selected fields of view (200×)/tissue section were assessed and a mean score was calculated for each section. All measurements are expressed as the mean score of each type of tissue.
Statistical analysis
For statistical analysis, commercial software was used.k Age and body weight were compared between the dogs with ICRPs (n = 15) and control dogs (5) with a Mann-Whitney U test. The activities of MMPs (pro–MMP-2, MMP-2, pro–MMP-9, and MMP-9) and serine proteases (SNBI) were compared among tissue type groups (inflamed and noninflamed colorectal mucosa from dogs with ICRPs, each n = 15; noninflamed colorectal mucosa from control dogs, 5) with a Kruskal-Wallis test followed by a posthoc Steel-Dwass test. Correlations between the activities of MMP-2 or MMP-9 and those of serine proteases (SNBI) were analyzed with the Spearman rank correlation test. Immunohistochemical scores for the inflamed colorectal mucosa samples from dogs with ICRP (n = 10) and colorectal mucosa tissue samples from control dogs (5) were compared with a Mann-Whitney U test. A value of P < 0.05 was considered significant.
Results
Fifteen MDs with ICRPs (1 sexually intact female, 5 spayed females, 4 sexually intact males, and 5 castrated males) were included in the study. The median age and weight of these dogs were 10 years (range, 7 to 12 years) and 5.3 kg (range, 3.5 to 7.7 kg), respectively. Histologically, inflamed lesions of all MDs with ICRPs had severe infiltrations of inflammatory cells composed mainly of neutrophils, lymphocytes, and macrophages. Five control dogs (2 spayed females and 3 castrated males) were included in the study. On the basis of histopathologic findings, 4 dogs had adenocarcinoma of the rectum and 1 dog had no apparent lesions. These dogs included 2 Toy Poodles, 1 West Highland White Terrier, 1 Shetland Sheepdog, and 1 MD. The median age and weight of these dogs were 11 years (range, 8 to 13 years) and 6.9 kg (range, 4.5 to 15.2 kg), respectively. Neither age nor weight differed significantly (P = 0.69 and P = 0.36, respectively) between the 2 groups of dogs.
In the gelatin zymography assays, gelatinolysis in samples appeared as clear bands in place of control bands (Figure 1). Activities of pro–MMP-2 and pro–MMP-9 were detected in most tissue samples regardless of group, whereas no activity of MMP-2 or MMP-9 was detected in the tissue samples from the control dogs (Figure 2). The activity of pro–MMP-2 was not significantly different among the 3 groups. On the other hand, the activity of pro–MMP-9 in the inflamed tissue samples (median activity, 4.88 AU; range, 1.18 to 7.56 AU) was significantly higher than that in the noninflamed samples (median activity, 0.61 AU; range, 0.09 to 6.27 AU; P < 0.001) or in the control tissue samples (median activity, 0.06 AU; range, 0 to 1.80 AU; P = 0.005). The activity of MMP-2 was significantly (P = 0.005) higher in the inflamed tissue samples (median activity, 0.02 AU; range, 0 to 0.15 AU) than in the noninflamed tissue samples (median activity, 0 AU; range, 0 to 0.02 AU). The activity of MMP-9 was significantly (P = 0.040) higher in the inflamed tissue samples (median activity, 0.03 AU; range, 0 to 0.78 AU) than in the noninflamed tissue samples (median activity, 0; range, 0 to 0.08).
Representative images of the results of gelatin zymography used to evaluate activities of MMP-2, MMP-9, and latent forms of MMP-2 and MMP-9 (pro–MMP-2 and pro–MMP-9) in inflamed (and noninflamed [not shown]) colorectal mucosa tissue samples obtained from MDs with ICRPs and in noninflamed colorectal mucosa tissue samples from dogs with non–ICRP-related large bowel diarrhea (controls). Tissue samples were collected endoscopically. The bands in lane 1 are the internal standards (pro–MMP-9, pro–MMP-2, and MMP-2 activities) used in each assay. Lanes 2, 3, and 4 represent pro–MMP-9 and pro–MMP-2 activities. Lanes 5 and 6 represent pro–MMP-9, pro–MMP-2, MMP-9 and MMP-2 activities. One tissue sample was run in each of lanes 2 through 6. Control samples in lanes 2 and 3 and inflamed tissue samples in lanes 4, 5, and 6 were derived from different dogs.
Citation: American Journal of Veterinary Research 81, 7; 10.2460/ajvr.81.7.572
Results of gelatin zymographic analyses for pro–MMP-2 (A), MMP-2 (B), pro–MMP-9 (C), and MMP-9 (D) activities in colorectal mucosa tissue samples obtained from 5 control dogs (Con) and 15 MDs dogs with ICRPs (both noninflamed [NI] and inflamed [I] tissues). One sample of tissue was analyzed for each dog. In each panel, a horizontal line indicates the median value for a sample gouping. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 81, 7; 10.2460/ajvr.81.7.572
In the serine protease zymography assays, the proteolytic activities in tissues samples followed a pattern similar to that observed in the gelatin zymography assays (Figure 3). Serine protease activities were detected in all tissue samples. Proteolytic activity was significantly (P = 0.006) higher in the inflamed tissue samples (median SNBI, 5.52 AU; range, 1.72 to 22.48 AU), compared with that in the control tissue samples (median SNBI, 0.47 AU; range, 0.19 to 2.72 AU [Figure 4]). Likewise, proteolytic activity was significantly (P = 0.039) higher in the noninflamed tissue samples (median SNBI, 3.16 AU; range, 0.87 to 22.73 AU), compared with that in the control tissue samples. However, there was no significant (P = 0.149) difference in SNBI between inflamed and noninflamed tissue samples.
Representative images of the results of zymography used to evaluate activities of serine proteases in inflamed (and noninflamed [not shown]) colorectal mucosa tissue samples obtained from MDs with ICRPs and in noninflamed colorectal mucosa tissue samples from dogs with non–ICRP-related large bowel diarrhea (controls). The band in lane 1 is an internal standard (plasmin activity). Lanes 2, 3, and 4 each show the activity of a single serine protease. Lanes 5 and 6 show the activities of multiple serine proteases. One tissue sample was run in each of lanes 2 through 6. Control samples in lanes 2 and 3 and inflamed tissue samples in lanes 4, 5, and 6 were derived from different dogs. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 81, 7; 10.2460/ajvr.81.7.572
Results of zymographic analyses of serine proteases in colorectal mucosa tissue samples obtained from 5 control dogs (Con) and 15 MDs dogs with ICRPs (both noninflamed [NI] and inflamed [I] tissues). One sample of tissue was analyzed for each dog. Because various serine proteases can be detected by serine protease zymography, the activities of serine proteases were measured as total proteolysis (SNBI) per lane and are reported as AU; values were calculated by comparing the intensity of the total activity to the intensity of the plasmin standard activity on each gel. In each panel, a horizontal line indicates the median value for a sample grouping.
Citation: American Journal of Veterinary Research 81, 7; 10.2460/ajvr.81.7.572
When the association of serine protease activities (ie, SNBI) with MMP-2 or MMP-9 activity was analyzed, a weak correlation was detected between SNBI and MMP-9 activity (rs = 0.34; P = 0.046). However, no correlation was detected between SNBI and MMP-2 activity (rs = 0.23; P = 0.177).
Immunohistochemical analyses revealed that MMP-9 was abundantly expressed in the inflamed tissue samples from MDs with ICRPs, especially in inflammatory cells such as neutrophils and macrophages. Expression of MMP-2 was detected in inflammatory cells, ECM, and fibroblasts in the granulomatous tissue of the lesions (Figure 5). The median immunohistochemical score for MMP-2 in the inflamed tissue samples from MDs with ICRPs was 2 (range, 1 to 2), whereas that score for the tissue samples from control dogs was 0 (range, 0 to 0; P < 0.001). Similarly, the median immunohistochemical score for MMP-9 in the inflamed tissue samples from MDs with ICRPs was 3 (range, 2 to 3), whereas that score for the tissue samples from control dogs was 1 (range, 0 to 1; P = 0.001).
Representative photomicrographs of immunohistochemical analyses of MMP-2 (A, B, E, F, I, and J) and MMP-9 (C, D, G, H, K, and L) expression in sections of inflamed colorectal mucosa tissue samples obtained from an MD with ICRPs and in noninflamed colorectal mucosa tissue samples obtained from a control dog. There was no staining for MMP-2 in the colorectal mucosa in the control dog (A and B). Expression of MMP-2 was detected in inflammatory cells, ECM, and fibroblasts (arrowhead) in the granulomatous lesion of an inflamed tissue section (E and F). A negative control for MMP-2 staining in an inflamed tissue section is shown (I and J). Staining for MMP-9 was slightly positive in the epithelium (white arrow) in a colorectal mucosa section obtained from the control dog (C and D). There was abundant expression of MMP-9 in the inflamed tissue, especially in inflammatory cells such as neutrophils and macrophages (black arrow [G and H]). A negative control for MMP-9 staining in an inflamed tissue section is shown (K and L). In each panel, bar = 50 mm. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 81, 7; 10.2460/ajvr.81.7.572
Discussion
In the study of the present report, we used zymographic methods to investigate the activities of MMPs and serine proteases in inflamed tissue samples obtained from MDs with ICRPs. The results of the study indicated that the activities of MMP-2, pro–MMP-9, and MMP-9 in inflamed tissue samples obtained from MDs with ICRPs were significantly higher than those in noninflamed tissue samples obtained from the same dogs. Also, the activities of serine proteases in both the inflamed and noninflamed tissue samples obtained from MDs with ICRPs were higher than those in tissue samples obtained from control dogs. These findings suggested that MMPs and serine proteases likely have important roles in the pathogenesis of ICRPs in MDs.
The activity of pro–MMP-9 was significantly increased in the inflamed tissue samples, compared with that in the control tissue samples, which was consistent with results of a previous study10 of humans with IBD. The median activity of pro–MMP-9 in the inflamed tissue samples obtained from MDs with ICRPs was approximately 8 times that in the noninflamed tissue samples obtained from the same dogs and approximately 80 times than that in the control tissue samples. Although these findings were similar to those observed in humans with IBD, the difference in median activity of pro–MMP-9 between the inflamed tissue samples obtained from MDs with ICRPs and control tissue samples was much larger; in humans with ulcerative colitis, the activity of pro– MMP-9 in inflamed mucosal samples is approximately 8 times that in control mucosal samples obtained from patients with colorectal carcinomas.10 In 1 study,14 pro–MMP-9 activity was identified in > 90% of mucosal samples obtained from dogs with chronic enteropathies, which was a higher percentage than that among samples from healthy control dogs; however, there was only an approximately 2-fold increase in median activity in the inflamed tissue samples. The reason for the notably greater difference in pro–M M P-9 activity between tissue samples from MDs with ICRPs and control dogs in the present study might be that the lesions of this disease contained abundant neutrophils and macrophages, which are the leading sources of pro–MMP-9.6,21 In the present study, the immunohistochemical analyses revealed that MMP-9 was abundantly expressed in neutrophils and macrophages in the inflamed tissue samples obtained from MDs with ICRPs, which corresponded with findings in inflamed tissue samples obtained from humans with IBD.10 In addition, it has been reported that the expression of MMP-9 is stimulated by inflammatory cytokines such as interleukin-1β and tumor necrosis factor-α,22 the expressions of which are reportedly upregulated in ICRPs of MDs.23
Although activities of pro–MMP-2 and pro–MMP-9 were detected in most colorectal mucosa tissue samples in the present study, active MMP-2 and MMP-9 were not detected in the control tissue samples. These results were consistent with those of previous studies9,14,17 of IBD in humans and chronic enteropathy in dogs, wherein no MMP-2 or MMP-9 activity was found in control tissues. Metalloproteinase-9 was detected in 60% of the inflamed and 20% of the noninflamed tissue samples obtained from the MDs with ICRPs in the present study; these detection rates were similar to those for inflamed and noninfamed tissue samples in a study9 of humans with IBD. In dogs with chronic enteropathies, MMP-9 activity has been identified in 5.6% of colonic mucosal samples,14 which is a detection rate lower than those determined in the present study and in the study9 of humans with IBD. This detection rate difference may possibly be attributed to the type of infltrative cells because neutrophils are not a main component of canine chronic enteropathy.14 These results have suggested that the activation of MMP-9 in the intestines does not commonly occur under physiologic conditions, whereas it contributes to intestinal inflammatory processes, especially neutrophilic inflammation. The activity of MMP-9 is strictly controlled at various stages, including the activation of the latent enzyme by several proteases, inhibition of MMPs by tissue inhibitors, and regulation of transcriptional levels.22 Under physiologic conditions, the intestinal mucosa has low-level pro–MMP-9 activity that likely functions in maintaining the turnover of the ECM, whereas abnormal activation of MMP-9 causes the destruction of the ECM through the enzyme's proteolytic function and enhances the recruitment of inflammatory cells, resulting in intestinal inflammation.24 The results of the present study indicated that ICRP lesions in MDs possess remarkable pro–MMP-9 and MMP-9 activities, which suggests that the expression and activation of MMP-9 might be important processes in the pathogenesis of ICRPs and may warrant investigation as possible targets for treatment interventions.
Targeting of MMP-9 appears to be a promising treatment for IBD because genetic or pharmacological suppression of MMP-9 in rats and mice with experimentally induced colitis alleviates colonic inflammation,25–28 although a recent clinical trial of an anti–MMP-9 antibody in humans with IBD failed to induce clinical remission.29,30 Another way to regulate the activation of MMPs is by targeting serine proteases that cleave the propeptide of pro–MMPs.6,11,12 Therefore, we investigated the serine protease activities in ICRPs in MDs. The results of the present study indicated that the activities of serine proteases were higher in the inflamed and noninflamed colorectal mucosa tissue samples obtained from MDs with ICRPs, compared with findings for colorectal mucosa tissue samples obtained from control dogs. A previous study5 in mice with experimentally induced colitis revealed apparently high serine protease activities in colonic lesions and identified correlations between the activities of MMPs and those of serine proteases, consistent with the finding of correlation of the activity of MMP-9 with the activities of serine proteases in the present study. On the other hand, the activity of MMP-2 had the highest association with the activities of serine proteases in the previous study,5 which was inconsistent with the finding of no correlation of the activity of MMP-2 with the activities of serine proteases in the present study. Recently, functional proteomic profiling of serine proteases in human IBD lesions has revealed that the activities of several serine proteases, such as cathepsin G and thrombin, are upregulated in patients with IBD, compared with findings for healthy controls.13 Although there are some discrepancies among the results of studies of IBD in humans, colitis in mice, and ICRPs in dogs, MMPs and serine proteases appear to interact with each other and to be associated with colorectal inflammation, as indicated by data obtained from mice with experimentally induced colitis.5 The results of the present study have suggested that serine proteases are possibly associated with ICRP-related disease in MDs through activation of MMP-9 or its inherent protease activity, thereby influencing inflammation, cell migration, and tissue damage.
In the present study, the activity of MMP-2 was higher in inflamed tissue samples than in noninflamed tissue samples obtained from MDs with ICRPs, although no significant difference in pro– MMP-2 activity between the 2 sample groups was detected. This finding was likely attributable to conversion of latent pro–MMP-2 into active MMP-2, as supported by findings in mice with experimentally induced colitis wherein pro–MMP-2 activity is not significantly different between tissue samples from mice with colitis and control mice, but MMP-2 activity is significantly higher in mice with colitis than in control mice.5 In the present study, immunohistochemical analyses revealed localization of MMP-2 expression in inflammatory cells, ECM, and fibroblasts in MDs with ICRPs. A relatively high level of MMP-2 activity has been postulated to be involved in tissue remodeling because MMP-2 is generally expressed in the ECM of the submucosa, epithelial cells, basement membrane, and fibroblasts.10
The present study had several limitations. First, the number of samples, especially in the control group, was small, and this sample size was possibly associated with decreased statistical power. For the control group, dogs with diseases other than ICRPs, such as adenocarcinoma of the rectum, were included. This approach enabled us to select age-matched control dogs but provided a small number of animals from which to collect colorectal mucosa tissue samples. Because the number of dogs with ICRPs was also small, further investigation of samples from a large number of dogs would be necessary to reinforce the study findings. Second, although the tissue samples collected from control dogs and the noninflamed tissue samples collected from MDs with ICRPs for zymography were macroscopically normal and located adjacent to histologically normal mucosa, the possibility of infiltration by tumor or inflammatory cells was not completely excluded, as suggested by the extremely high pro–MMP-9 and serine proteases activities in the noninflamed colorectal mucosa tissue obtained from an MD with ICRPs. In addition, the ranges of MMPs and serine protease activities in the inflamed tissue samples obtained from MDs with ICRPs were wide, possibly because of variation in inflammation severity. Inflammation severity was not objectively assessed in the present study, largely because ICRP-related lesions are heterogeneous and evaluation of the inflammation severity is fairly difficult. However, dense infiltrates of inflammatory cells, such as neutrophils, lymphocytes and macrophages, are typical of ICRP-related disease in dogs, and such infiltrates were evident in inflamed colorectal mucosa tissue samples obtained from all MDs with ICRPs in the present study. Finally, because the serine protease zymography method used in the study detected various types of serine proteases, we analyzed the total activities of serine proteases. Therefore, the activities of specific serine proteases, such as plasmin, neutrophil elastase, urokinase, trypsin, or chymotrypsin, were not confirmed.5,9 Moreover, we used the same amounts of samples for all groups in the zymography assays to compare the activities among groups, resulting in the presence of undifferentiated bands in some samples and difficulties in accurate calculation of activities in each band, especially those of pro–MMP-9 and MMP-9. It was difficult to determine the appropriate sample amount to be loaded on the zymography assays because pro–MMP-9 and MMP-9 activities were considerably different between ICRP-related lesions and control tissues. Further studies are needed to confirm the activities of specific serine proteases and MMP-9 activities in colorectal mucosa tissue obtained from MDs with ICRPs. Despite these limitations, results of the present study have suggested that MMP-2, MMP-9, and serine proteases may have important roles in the pathogenesis of ICRPs in MDs, given that these proteases are known to be essential for ECM degradation, inflammatory cell migration, and inflammatory responses within the lesions of various inflammatory disorders.
Acknowledgments
Supported by JSPS KAKENHI Grant No. JP18J21178. Funding sources did not have any involvement in the study design, data analysis and interpretation, or writing and publication of the manuscript.
The authors declare that there were no conflicts of interest.
The authors thank Dr. Yumiko Kagawa for interpretation of the histopathologic findings.
ABBREVIATIONS
| AU | Arbitrary units |
| ECM | Extracellular matrix |
| IBD | Inflammatory bowel disease |
| ICRP | Inflammatory colorectal polyp |
| MD | Miniature Dachshund |
| MMP | Matrix metalloproteinase |
| SNBI | Sum net band intensity |
Footnotes
Complete Mini EDTA-free Protease Inhibitor Cocktail, Roche Diagnostics GmbH, Mannheim, Germany.
Nacalai Tesque, Kyoto, Japan.
Cosmo Bio Co Ltd, Tokyo, Japan.
ImageJ software, National Institutes of Health, Bethesda, Md.
Life Laboratory, Yamagata, Japan.
Biovision, Milpitas, Calif.
Nichirei Biosciences, Tokyo, Japan.
Proteintech, Chicago, Ill.
Abcam, Cambridge, England.
Santa Cruz Biotechnology, Dallas, Tex.
JMP Pro, version 14.0, SAS Institute Inc, Cary, NC.
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