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Keywords:

  • MMP;
  • TIMP;
  • Crohn's disease;
  • diverticulitis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Background:

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), are expressed in the gastrointestinal tract by different cellular types. Nevertheless, the imbalance between MMPs and TIMPs plays an important role in the physiopathology of diverse intestinal inflammatory processes.

Methods:

An immunohistochemical study was performed using tissue arrays and specific antibodies against MMPs -1, -2, -7, -9, -11, -13, -14, and TIMPs -1, -2 and -3. Immunohistochemical staining of intestinal samples from surgical interventions from 30 patients with complicated Crohn's disease (CD) and 25 patients with diverticulitis were performed at the inflamed mucosa and in adjacent noninflamed mucosa. A reverse-transcription polymerase chain reaction (RT-PCR) analysis was performed to confirm the results obtained by immunohistochemistry. In addition, western blot experiments were carried out.

Results:

CD inflamed mucosa showed higher global expression of MMP-2, MMP-9, and MMP-13 than diverticulitis inflamed mucosa. However, inflamed and noninflamed diverticulitis mucosal samples showed higher global expression of MMP-1, TIMP-1, and 3 than the CD samples. Epithelial cells of inflamed mucosa showed higher expression of MMP-2, 9, and 13 in CD than diverticulitis. However, the latter showed higher expression of TIMP-1. Similar differences for fibroblast-like cells and mononuclear inflammatory cells were found. CD samples presented an increased expression of MMPs and a decreased expression of TIMPs compared to diverticulitis.

Conclusions:

These results indicate a differential pattern of expression of MMPs and TIMPs in CD and diverticulitis and the necessity to study the potential role of MMP inhibitors as new protective agents in both diseases. (Inflamm Bowel Dis 2011;)

Crohn's disease (CD) is a chronic inflammatory disorder of the gastrointestinal tract characterized by relapses and remissions. It is characterized by chronic, multifocal transmural inflammation potential affecting the whole gastrointestinal lining, particularly in the ileocolonic region. Although the etiology is unknown, current understanding suggests that sustained immune infiltration manifests as an aberrant reaction to luminal bacteria in genetically susceptible individuals.1 Chronic inflammation is characterized by an inflammatory cell infiltrate associated with changes in the architecture, which consists of an increased turnover of extracellular matrix (ECM) components.2 The disturbance of the balance between synthesis and degradation of ECM components may result in the modification and degeneration of the normal bowel wall architecture, leading to a progressive organ destruction, as seen, for example, in the process of ulcer formation,3 or excessive deposition of collagen, resulting in fibrosis2, 4. A number of studies suggest that matrix metalloproteinases (MMPs) are the most important group of proteolitic enzymes responsible for the breakdown of ECM in inflammatory bowel diseases.5–11 In addition, studies in animal models have shown therapeutic results utilizing specific MMP inhibitors.12, 13

Diverticular disease of the colon is very common in the Western world, especially at older age. In most patients the clinical picture of the diverticular colonic disease is a quiescent or even asymptomatic disease. However, some patients develop acute or chronic inflammation expressed as diverticulitis, which comprises a wide spectrum of clinical presentation, including complications such as perforation. Complicated diverticular disease, or diverticulitis, is characterized by profound structural changes in the colonic tissue, varying from tissue disruption to fibrosis; it has been hypothesized that an unbalanced expression of MMPs and their inhibitors (TIMPs) protein expression in the bowel wall might, at least in part, mediate this process.14 However, there is limited knowledge about the role of MMPs in the pathogenesis of diverticulitis. A recent article has reported that altered concentrations of MMPs and TIMPs may explain the structural changes found in diverticulitis.14

The human MMP family currently consists of 23 members of homologous zinc-dependent endopeptidases, which can be divided into eight structural classes, or based on their substrate specificity (Table 1) and primary structure, into the more familiar subgroups of collagenases (MMP-1, 8, and 13), gelatinases (MMP-2 and 9), stromelysins (MMP-3, 10, 11), membrane-associated MMPs (MMP-14, 15, 16, 17, 23, 24, 25), and other novel MMPs.15, 16 They play an essential role in the degradation of the stromal connective tissue and basement membrane components. In addition, MMPs are able to impact in vivo cell behavior as a consequence of their ability to cleave growth factors, cell surface receptors, cell adhesion molecules, and chemokines/cytoquines.17–19 MMPs also regulate angiogenesis, positively through their ability to mobilize or activate proangiogenic factors,20 and negatively via generation of angiogenesis inhibitors, such as angiostatin and endostatin, cleaved from large protein precursors.21 MMPs are synthesized as inactive zymogens, which are then activated predominantly pericellularly by either other MMPs or by serine proteases. MMPs' activity is specifically inhibited by the so-called tissue inhibitors of metalloproteases (TIMPs). Currently, four different TIMPs are known to exist: TIMPs 1, 2, 3, and 4. TIMP-1, -2, and -4 are present in soluble forms, while TIMP-3 is tightly bound to the matrix.22 Numerous studies have indicated that, independently of MMP inhibition, TIMPs are multifactorial proteins involved not only in tissue remodeling and wound healing, but also in many other physiological and pathological processes such as angiogenesis, steroidogenesis, hematopoiesis, cell growth, and cell survival.22

Table 1. Human Matrix Metalloproteinases
MMP NumberCommon NameMolecular Weight kDaSubstrate
  • *

    Zymogen molecular weight, A active form molecular weight.

MMP-1Collagenase-157*Collagen types I, II, III, VII, X, proteoglycans
Interstitial collagenase47 A 
MMP-2Gelatinase A72*Gelatin, Collagen types IV, V, VII, IX, X
66 AFibronectin, Elastin
MMP-3Stromelysin-160*Fibronectin, Laminin, Elastin, Proteoglycans, Gelatin, Collagen types IV, V and IX
52 A
MMP-7Matrilysin28*Gelatin, Fibronectin, Laminin, Elastin, Collagen type IV
19 A
MMP-8Neutrophil-type collagenase85*Collagen types I, II, III, VII, VIII, X, Fibronectin and Proteoglycans
Collagenase-264 A
MMP-9Gelatinase B92*Gelatin, Collagen types IV, V, VII,X, Fibronectin, Elastin and Proteoglycans
86 A
MMP-10Stromelysin-253*Fibronectin, Casein, Gelatin, Collagen types I, III, IV and V
47 A
MMP-11Stromelysin-360*Collagen types IV, V, IX, X, Laminin, Elastin, Fibronectin and Proteoglycans
47 A
MMP-12Macrophage Elastase54*Elastin
45 A
MMP-13Collagenase-360*Collagene I, II, III, VII, VIII and X, Tenascin, Gelatin
48 A
MMP-14Membrane type-1 MMP66*Pro-MMP2, Collagen types I, II, III, Gelatin, Fibronectin, Vitronectin, Laminin
MT1-MMP54 A
MMP-15Membrane type-2 MMP76Pro-MMP2, Gelatin, Fibronectin, Vitronectin, Laminin
MT2-MMP
MMP-16Membrane type-3 MMP65*Pro-MMP-2, Collagen type III, Fibronectin, Gelatin
MT3-MMP63 A
MMP-17Membrane type-4 MMP65*Pro-MMP-2, Fibrin/fibrinogen, Gelatin
MT4-MMP63 A
MMP-19RASI-159Native type IV collagen, Gelatin, Laminin, Fibronectin
MMP-20Enamelysin56Amelogenin, Aggrecan
MMP-23CA-MMP44Gelatin
MMP-24Membrane type-5 MMP73Pro-MMP2, Fibronectin
MT5-MMPProteoglycans
 Gelatin
MMP-25Membrane type-6 MMP62Pro-MMP2, Pro-MMp9, Native type IV collagen
MT6-MMPGelatin, Fibronectin, Proteoglycans, Laminin-1
LeukolysinFibrin/fibrinogen
MMP-26Matrilysin-229Pro-MMP9, Native type IV collagen
Gelatin
Fibronectin
Fibrin/fibrinogen
MMP-2759
MMP-28Epilysin59

The aim of this study was to investigate the differential expression of several MMPs and TIMPs in CD and diverticulitis at the mucosal level.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Patient Selection, Characteristics, and Tissue Specimen Handling

This study included 30 patients with CD and 25 with diverticulitis. All patients underwent surgery for disease complications between 1990 and 2003. Characteristics of included patients are listed in Table 2.

Table 2. Clinical Characteristics and Treatment of Crohn's Disease and Diverticulitis Patients at the Time of Study Inclusion
 Crohn's DiseaseDiverticulitis
Cases3025
Age, years (mean ± SD)36.9 ± 13.364.3 ± 13.2
Sex females:males12:1813:12
Phenotype8 (26.7%) stenosing 
22 (73.3%) penetrating
Type of surgery (urgent: programmed)0.51250.55
Surgery indication  
 Appendicitis suspicion2 
 Abscess82
 Intestinal perforation39
 Stenosis/obstruction104
 Fistulization41
 No response2 
 Perianal disease1 
 Chronic pain 7
 Cancer 2
Current treatment  
 No medication5 
 5-ASA21 
 Salazopyrin2 
 Budesonide2 
 Corticoids18 
 Azathioprine8 
 Infliximab1 
 Antibiotics 25

Tissue Microarrays and Immunohistochemistry

Tissue samples were obtained at the time of surgery. They were fixed immediately in 10% buffered formalin overnight. Fixed tissue samples were dehydrated in ethanol, cleared in xylene, and embedded in paraffin blocks. Representative histological inflammatory and noninflammatory areas were defined on hematoxylin and eosin (H&E)-stained sections and marked on the slide. Tissue array (TMA) blocks were performed as described by Parker et al.23 A total of four cores were used for each case. Two of these cores in each case corresponded to the more inflamed mucosal area, and the other two cores corresponded to the adjacent noninflamed mucosal area.

Serial 5-μm sections of the high-density TMA blocks were consecutively cut with a microtome (Leica Microsystems, Wetzlar, Germany) and transferred to adhesive-coated slides. One section from each tissue array block was stained with H&E, and these slides were then reviewed to confirm that the sample was representative of the original tissue. Immunohistochemistry was done on these sections using a TechMate TM50 autostainer (Dako, Glostrup, Denmark). Antibodies for MMPs and TIMPs were obtained from Neomarker (Lab Vision, Fremont, CA). The dilution for each antibody was established based on negative and positive controls (1/50 for MMP-2, -7, -14, and TIMP-2; 1/100 for MMP-9, -13, TIMP-1, and -3; and 1/200 for MMP-1, -11). The negative control was DakoCytomation mouse serum diluted to the same mouse IgG concentration as the primary antibody. On the other hand, we also used antibodies against cytokeratins (AE1-AE3, Dako 1/1) and vimentin (Dako 1/100) to distinguish fibroblasts-like cells from stromal cells. All the dilutions were made in Antibody Diluent (Dako) and incubated for 30 minutes at room temperature.

Tissue sections were deparaffinized in xylene and then rehydrated in graded concentrations of ethyl alcohol and then water. To enhance antigen retrieval only for some antibodies, TMA sections were microwave-treated in a H2800 Microwave Processor (EBSciences, East Granby, CT) in citrate buffer (Target Retrieval Solution; Dako) at 99°C for 16 minutes. Endogenous peroxidase activity was blocked by incubating the slides in peroxidase-blocking solution (Dako) for 5 minutes. The EnVisionDetection Kit (Dako) was used as the reactivity detection system. Sections were counterstained with hematoxylin, dehydrated with ethanol, and permanently coverslipped.

Image Analysis

For each antibody preparation studied, the location of immunoreactivity, percentage of reactive area, and intensity were determined. All the cases were semiquantified for each protein-stained area. An image analysis system with the Olympus BX51 microscope and soft analysis (analySIS, Soft imaging system, Münster, Germany) were used as follows. There were different optical thresholds for both stains. Each core was scanned with a 400× power objective in two fields per core. Fields were selected searching for the protein-reactive areas. The computer program selected and traced a line around antibody-reactive areas (higher optical threshold: red spots), with the remaining, nonstained areas (hematoxylin-stained tissue with lower optical threshold) standing out as a blue background. Any field had an area ratio of stained (red) versus nonstained (blue). A final area ratio was obtained after averaging two fields. To evaluate immunostaining intensity we used a numeric score ranging from 0 to 3, reflecting the intensity as follows: 0, no reactivity; 1, weak reactivity; 2, moderate reactivity; and 3, intense reactivity. Using an Excel spreadsheet, the mean score was obtained by multiplying the intensity score (I) by the percentage of reactivity area (PA) and the results were added together (total score: I × PA). This overall score was then averaged with the number of cores that were done for each patient. If there was no tissue in a particular core, then no score was given. In addition, for each sample the mean score of two core biopsy samples was calculated. This scoring evaluation was based on a global evaluation of staining areas corresponding to epithelial cells as well as to fibroblast-like cells and mononuclear inflammatory cells (MICs). Nevertheless, in the present work we also evaluated the immunohistochemical staining by each cellular type. We distinguished epithelial cells from stromal cells. Fibroblast-like cells are spindle cells, whereas mononuclear inflammatory cells are round cells. On the other hand, while epithelial cells are arranged forming either acinar or trabecullar pattern, stromal cells are spread. Moreover, we used two markers to distinguish fibroblast-like cells from epithelial cells: cytokeratins and vimentin, as it described above.

Furthermore, whole-tissue sections from specimen blocks from a subset of 10 cases were compared with the corresponding TMA discs, regarding each MMP and TIMP expression. Those cases were selected randomly, and the obtained clinicopathological data were very similar to those from the whole series. Each whole-tissue section was scanned with a 400× power lens in 10 different fields. Fields were selected searching for the protein-stained areas, such as described above.

Statistical Analysis

Differences in percentages were calculated with the chi-square test. Immunostaining score values for each protein were expressed as a mean (range). Comparison of immunostaining values between groups was made with the Mann–Whitney or Kruskall–Wallis tests. Statistical results were corrected applying Bonferroni's correction. The SPSS 17.0 program (Chicago, IL) was used for all calculations.

Quantitative Reverse-transcription Polymerase Chain Reaction (RT-PCR)

Total RNA was isolated from formalin-fixed, paraffin-embedded tissue blocks using the Nucleospin FFPE RNA Kit (Macherey-Nagel, Düren, Germany), including DNase treatment. The integrity of the eluted total RNA was checked by agarose gel electrophoresis and the RNA concentration was determined spectrophotometrically (NanoDrop Technologies, Wilmington, NC). First-strand cDNA was made using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cheshire, UK) following the manufacturer's instructions. The reverse transcription step was carried out using the following program: 25°C for 10 minutes, 37°C for 120 minutes, and 85°C for 5 minutes. Expression of MMP-1, -2, -9, -13, TIMP-1, -3, and β-actin expression levels were assessed by real-time PCR using an ABI Prism 7900 HT thermocycler (Applied Biosystems) and the Fast SYBR Green Master Mix (Applied Biosystems) with the following cycling conditions: 95°C for 20 seconds and 40 cycles of 95°C for 1 second and 60°C for 20 seconds.

Protein Extraction

Five FFPE tissue sections (50 μm each) were deparaffinized by adding 0.5 ml xylene and incubated 8 minutes at 60°C. Xylene was removed and sections were rehydrated in successive washes in 100%, 96%, and 70% ethanol and water. After centrifugation the water was removed and the residuum dried in a hood for 2–3 minutes. Two hundred μL of 20 mM Tris-HCl buffer (pH 9) containing 2% sodium dodecyl sulfate (SDS) was then added to the dewaxed FFPE tissues sections, followed by heating at 100°C on a heat block (Fisher Scientific; Pittsburgh, PA) for 20 minutes, then incubation at 60°C in an Eppendorf Thermomixer (shaking at 650 rpm) for 2 hours.24 Protein concentrations were determined using the Bradford Method with bovine serum albumin (BSA) as standard (Sigma, St. Louis, MO). The samples were treated according to the manufacturer's “microwell” protocol and the absorbance was read at 495 nm on a plate reader (Bio-Rad, Munich, Germany).

Western Blot

Protein samples (40 μg/lane) were separated by SDS-PAGE using 10% acrylamide resolving and 4% stacking gels. After electrophoresis, the proteins were blotted from the SDS-PAGE onto 0.45 μm nitrocellulose membranes (BioRad Laboratories, Hercules, CA). Nonspecific binding sites were blocked by incubating the nitrocellulose membranes for 1 hour with 5% nonfat milk and Tris-buffered saline containing 0.05% Tween-20 (TBS-T). The membranes were then washed twice with TBS-T and incubated overnight with monoclonal antibodies antihuman MMP-1, -9 (1/500), MMP-2 (1/50), MMP-13 (1/1000), β-actin (1/700) (Neomarkers, Lab Vision) in 5% nonfat milk and TBS-T. The membranes were subsequently washed with TBS-T and incubated for 1 hour with Protein A-peroxidase (Sigma, cat. no. P8651) in 5% nonfat milk and TBS-T. Protein bands were detected using the Immobilon Western Chemiluminescent HRP substrate (Millipore, Bedford, MA) with subsequent exposure to x-ray film.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Immunohistochemical analyses of intestinal samples from surgical interventions from 30 patients with CD, 25 patients with diverticulitis were performed on TMAs. Minimal internal variance of score data between duplicate tissue cores from the same patients was detected in the tissue arrays, showing a high agreement for each protein (r > 0.95 and P < 0.0001, for each protein). In the validation study there was total concordance in the global expression, as well as in the intensity of immunostaining, for each MMP and TIMP between TMA cases and the corresponding whole-tissue sections. In addition, there were highly significant correlations in the immunostaining scores between these two paired sets (r > 0.90 and P < 0.0001, for each protein).

Figure 1 shows representative example of the expression of MMPs and TIMPs in CD tissue samples and in diverticulitis tissue samples. Immunostaining of these proteins shows a cytoplasmic localization in all positive cases.

thumbnail image

Figure 1. 2Representative examples of the expression of MMPs and TIMPS by Crohn's disease and diverticulitis. (A) 200×. MMP-1 expression in CD. (B) 200×. MMP-1 expression in diverticulitis. (C) 200×. MMP-2 expression in CD. (D) 200×. MMP-2 expression in diverticulitis. (E) 200×. MMP-9 expression in CD. (F) 200×. MMP-9 expression in diverticulitis. (G) 200×. MMP-13 expression in CD. (H) 200×. MMP-13 expression in diverticulitis. (I) 200×. TIMP-1 expression in CD. (J) 200×. TIMP-1 expression in diverticulitis. (K) 200×. TIMP-3 expression in CD. (L) 200×. TIMP-3 expression in diverticulitis. (M,N) Examples of selected areas used for quantification. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Table 3 shows the global value (score values) of the immunostaining for each protein evaluated in CD and diverticulitis cases, also at inflamed mucosa as at adjacent noninflamed mucosa. We found significantly higher expression of MMP-2 and -13 in inflamed mucosa than in noninflamed mucosa from patients with CD. However, inflamed mucosa showed a lower expression of TIMP-1 and -3 than adjacent noninflamed mucosa from patients with diverticulitis.

Table 3. Global Expression (Score Values) of Metalloproteases (MMPs) and Their Inhibitors (TIMPs) in Crohn's Disease and Diverticulutis
FactorInflamed Mucosa SamplesNoninflamed Mucosa Samples
Crohn's Disease (n = 30) Median [Range]Diverticulitis (n = 25) Median [Range]P-valueCrohn's Disease (n = 30) Median [Range]Diverticulitis (n = 25) Median [Range]P-valuea
  • a

    Mann-Whitney test.

  • *

    P < 0.05,

  • **

    P < 0.01 vs. paired set of noninflamed mucosa samples (Wilcoxon test).

  • Samples on tissue sections were either insufficient or lost for analysis in one case for MMP1, MMP2, MMP9, MMP11, MMP13, MMP14, TIMP1, and TIMP2, two cases for MMP7, and three cases for TIMP3 for the inflamed mucosa in patient with Crohn's disease. The same occurs with inflamed mucosa of patients with diverticulitis in 11 cases for MMP1, 10 cases for MMP2 and MMP7, nine cases for MMP9 and MMP11, six cases for MMP13, eight cases for MMP14, seven cases for TIMP1 and TIMP2, 12 cases for TIMP3. Samples on tissue sections were either insufficient or lost for analysis in two cases for MMP1, one case for MMP2, MMP7, MMP9, MMP11, MMP13, MMP14, TIMP1, and TIMP2, and three cases for TIMP3 for the noninflamed mucosa in patient with Crohn's disease. The same occurs with noninflamed mucosa of patients with diverticulitis in four cases for MMP1, two cases for MMP2, MMP9, and MMP11, three cases for MMP7, one case for MMP14 and TIMP2, and six cases for TIMP-3. The values shown correspond to the total of cases analyzed for each protein.

MMP-150.8 [0–143.1]**131.5 [0–176.3]0.00644.1 [0–139.4]129.66 [0–258.49]<0.000
MMP-232.2 [0–90.4]**0 [0–0]0.0010 [0–50.1]0 [0–0]0.06
MMP-744.2 [0–101.2]0 [0–175.5]0.4335.1 [0–109.8]0 [0–157.4]0.58
MMP-943.8 [0–104.4]0 [0–28.28]<0.00040.2 [0–116.7]0 [0–0]<0.000
MMP-11127.5 [0–213.8]62.5 [0–222.7]0.05471.8 [0–169.1]52.7 [0–209.4]0.15
MMP-130 [0–44.9]*0 [0–0]0.0000 [0–47.61]0 [0–0]0.056
MMP-140 [0–95.1]21.3 [0–133.3]0.320 [0–53.2]44.1 [0–110.4]0.001
TIMP-10 [0–113.9]44.5 [0–142.4]*0.0010 [0–56.9]66.1 [0–151.6]<0.000
TIMP-20 [0–75.8]*0 [0–73.5]0.080 [0–34.7]0 [0–29.6]0.75
TIMP-30 [0–45.2]43.2 [0–113.4]*0.0030 [0–52.3]69.9 [0–139.8]<0.000

When we compared the global expression of MMPs and TIMPs between inflamed mucosa of CD patients and diverticulitis patients, our results showed significant differences (Table 3). Inflamed mucosa of CD patients showed higher expression of MMP-2, MMP-9, and MMP-13 than inflamed mucosa of diverticulitis patients. Although the median of the CD group is 0 for MMP13, 14 samples of 30 were positive (range: 27.0–44.9) and none of the 25 samples of diverticulitis were positive. Therefore, the expression of MMP13 was significantly higher in inflamed mucosa of CD patients than inflamed mucosa of diverticulitis patients. However, inflamed mucosa of diverticulitis patients showed higher expression of MMP-1, TIMP-1, and 3 than inflamed mucosa of CD patients. Similar differences for these protein expressions were found between noninflamed mucosa of CD and of diverticulitis.

The results obtained by immunohistochemistry were confirmed by quantitative RT-PCR. These results are shown in Figure 2.

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Figure 2. Representative results of MMPs and TIMPs expression by quantitative RT-PCR. (A) Expression of MMPs statistically significant by immunohistochemistry in inflamed mucosa of patients with CD and diverticulitis. **P < 0.0001 and *P < 0.05. (B) Expression of TIMPs statistically significant by immunohistochemistry in inflamed and adjacent noninflamed mucosa of patients with diverticulitis. P < 0.0001.

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To assess MMPs activity and confirm the results obtained by immunohistochemistry and RT-PCR at the protein level, proteins were isolated from the same tissue samples that were used for the immunostaining and mRNA extraction. Using specific antibodies against MMPs a distinction between the latent pro-form and the active form of the MMPs was possible. Results of western blots are summarized in Figure 3. For MMP-2, -9, and -13 both expression of proMMP and active MMP were higher in CD inflamed mucosal samples compared to diverticulitis inflamed mucosal samples. Furthermore, for MMP-1 a higher expression of the active protein could be observed in diverticulitis inflamed mucosal samples.

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Figure 3. Expression of latent and active form of MMPs analyzed by western blot. Protein samples were separated by polyacrylamide gel electrophoresis and expression of MMP proteins was visualized using specific antibodies. β-Actin was used as internal loading control. Inactive (latent) and active forms of MMP proteins are indicated with arrows.

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When we analyzed the relationship between the medication and MMPs/TIMPs expression, we found no significant results (data not shown). Furthermore, we found no significant relationships between the different phenotypes of disease and MMPs/TIMPs expression (data not shown).

Tables 4, 5, and 6 show the percentages of expression of MMPs and TIMPs by each cellular type corresponding to the different pathologies. As can be seen in Table 4, we found that epithelial cells from inflamed samples of CD show significantly higher percentage of expression of MMP-1, 2, and 13 than epithelial cells from paired noninflamed mucosal samples. Similar differences in protein expressions were found between these two paired set of samples from CD protein expression by fibroblast-like cells (MMP-1, 2, 7, and 9) and for MICs (MMP-1, 2, and 9). With regard to the expression of MMPs or TIMPs by epithelial cells, MICs and fibroblast-like cells from patients with diverticulitis, there were no significant differences between inflamed samples and noninflamed paired samples.

Table 4. Expression of Metalloproteases (MMPs) and Their Inhibitors (TIMPs) by Epithelial Cells in Crohn's Disease and Diverticulitis
 Epithelial Cells in Inflamed MucosaEpithelial Cells in Noninflamed Mucosa
Crohn's DiseaseDiverticulitisPCrohn's DiseaseDiverticulitisP
No. (%)No. (%)No. (%)No. (%)
  • *

    P < 0.01,

  • **

    P < 0.001 vs. paired set of noninflamed mucosa samples (chi-square test).

MMP-128 (96.6)**13 (92.9)118 (64.3)18 (85.7)0.11
MMP-216 (55.2)*0 (0)<0.004 (13.8)0 (0)0.12
MMP-721 (75)6 (40)0.05316 (55.2)7 (31.8)0.16
MMP-923 (79.3)2 (12.5)<0.0020 (69)0 (0)<0.00
MMP-1125 (86.2)13 (81.3)0.6824 (82.8)15 (65.2)0.25
MMP-1314 (48.3)*0 (0)<0.004 (13.8)0 (0)0.11
MMP-1413 (44.8)9 (52.9)0.827 (24.1)17 (70.8)0.002
TIMP-112 (41.4)15 (83.3)0.00611 (37.9)19 (76)0.01
TIMP-28 (27.6)1 (5.6)0.123 (10.3)2 (8.3)1
TIMP-38 (29.6)9 (64.3)0.077 (25.9)14 (73.7)0.004
Table 5. Expression of Metalloproteases (MMPs) and Their Inhibitors (TIMPs) by Mononuclear Inflammatory Cells in Crohn's Disease and Diverticulitis
 Mononuclear Inflammatory Cells in Inflamed MucosaMononuclear Inflammatory Cells in Noninflamed Mucosa
Crohn's DiseaseDiverticulitisPCrohn's DiseaseDiverticulitisP
No. (%)No. (%)No. (%)No. (%)
  • *

    P < 0.01,

  • **

    P < 0.001 vs. paired set of noninflamed mucosa samples (chi-square test). n.s., not significant.

MMP-125 (86.2)**13 (92.9)13 (10.7)15 (71.4)<0.000
MMP-212 (41.2)*0 (0)0.0032 (6.9)0 (0)0.49
MMP-714 (50)6 (40)0.764 (13.8)6 (27.3)0.39
MMP-917 (58.6)**0 (0)<0.0000 (0)0 (0)n.s.
MMP-1125 (86.2)11 (68.8)0.2411 (37.9)10 (43.5)0.9
MMP-136 (20.7)0 (0)0.061 (3.4)0 (0)1
MMP-144 (13.8)7 (41.2)0.071 (3.4)12 (50)<0.000
TIMP-16 (20.7)11 (61.1)0.014 (13.8)13 (52)0.007
TIMP-20 (0)0 (0)n.s0 (0)0 (0)n.s.
TIMP-35 (18.5)8 (57.1)0.031 (3.7)11 (57.9)<0.000
Table 6. Expression of Metalloproteases (MMPs) and Their Inhibitors (TIMPs) by Fibroblasts in Crohn's Disease and Diverticulitis
 Fibroblasts In Inflamed MucosaFibroblasts In Noninflamed Mucosa
Crohn's DiseaseDiverticulitisPCrohn's DiseaseDiverticulitisP
No. (%)No. (%)No. (%)No. (%)
  • *

    P < 0.01,

  • **

    P < 0.001 vs. paired set of noninflamed mucosa samples (chi-square test) n.s., not significant.

MMP-122 (75.9)**10 (71.4)12 (7.1)7 (33.3)0.02
MMP-211 (37.9)*0 (0)0.0081 (3.4)0 (0)1
MMP-712 (42.9)*6 (40)12 (6.9)2 (9.1)1
MMP-917 (58.6)**0 (0)<0.0000 (0)0(0)n.s.
MMP-1124 (82.2)12 (75)0.714 (48.3)9 (39.1)0.70
MMP-136 (20.7)0 (0)0.060 (0)0 (0)n.s.
MMP-144 (13.8)5 (29.4)0.250 (0)2 (8.3)0.2
TIMP-10 (0)3 (16.7)0.051 (3.4)6 (24)0.04
TIMP-21 (3.4)0 (0)10 (0)0 (0)n.s.
TIMP-31 (3.7)8 (57.1)<0.0000 (0)8 (42.1)<0.000

When we compared the expression of MMPs and TIMPs by each cellular type, between inflamed mucosa of CD and inflamed mucosa of diverticulitis, our results showed several significant differences (Tables 4, 5, and 6). Epithelial cells from inflamed mucosa of CD showed higher percentages of expression of MMP-2, 9, and 13 than inflamed mucosa of diverticulitis. However, inflamed mucosa of diverticulitis showed higher expression of TIMP-1 than inflamed mucosa of CD. As Tables 5 and 6 show, similar differences for MICs and fibroblast-like cells were found in all of these comparisons. In general, we found greater percentages of expression of MMPs and smaller percentages of expression of TIMPs in CD samples than in diverticulitis samples in mucosa inflamed samples as in noninflamed samples.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The present study investigated the mucosal expression of seven MMPs and three TIMPs in both complicated CD and diverticulitis. Therefore, mucosal samples studied belong to cases with the most advanced stages of these processes, in which surgical treatment was necessarily applied due to relevant architecture alteration such as: perforation, fibrotic strictures, or fistulae. However, we found different expressions of these proteins between these two pathologies, suggesting different pathophysiological mechanisms. Comparing both processes, CD cases showed higher expression of MMP-2, MMP-9, and MMP-13 than diverticulitis cases, whereas the latter process showed higher expression of MMP-1, TIMP-1, and TIMP-3.

Our results are in accordance with a previous report indicating high expression of MMPs in inflammatory bowel disease. MMP-2 (gelatinase A) has a special capacity to degrade the type IV collagen found in basement membranes.25 Nevertheless, recently a protective role for MMP-2 was demonstrated in experimental colitis.26 In fact, there are data showing that MMPs and TIMPs are not only involved in tissue damage but also in intestinal wound healing, reepitheliazation, myofibroblast and immune cell migration, scar formation, fibrogenesis, and neovascularization in the intestine.27–29

On the other hand, our results indicate that diverticulitis cases have higher expression of MMP-1, TIMP-1, and TIMP-3 than CD cases. It is of note that MMP-1 and TIMP-1 were found increased in intestinal segments affected by diverticulitis and distributed throughout the entire bowel wall, which may explain the structural changes.14 MMP-1 (collagenase-1) is the most ubiquitously expressed of the interstitial collagenases. MMP-1 cleaves several components of the ECM, including collagen of types I, II, III, VII, VIII, and IX, aggrecan, as well as serin proteinase inhibitors, and α2 macroglobulin.30 However, it is of note that we found no different MMP-2 levels between inflamed and noninflamed samples from diverticulitis, whereas other authors found increased MMP-2 levels in complicated diverticular disease. We think that both different design and methodological aspects may explain these apparently different results. These include patient populations with different phenotype disease, different antibodies used for MMP/TIMP expressions, or different evaluation of the location of MMP/TIMP expressions (muscularis propria or mucosal and submucosal samples). With regard to the latter aspect, the findings of Mimura et al31 are noteworthy in that TIMP-1 and 2 expressions were significantly higher in the muscular layer of complicated diverticulosis than controls. These findings may indicate that both TIMP-1 and TIMP-2 overexpression in the muscular layer affects the turnover of extracellular matrix, as a secondary event, resulting in the formation of colonic diverticula. On the other hand, when the expression of MMPs of TIMPs by epithelial cells and MICs were compared in inflamed and noninflamed paired samples from patients with diverticulitis, no significant differences were found. However, we found a higher percentage of expression of MMP-1, 7, and 11 (stromalysin-3) by fibroblast-like cells from inflamed samples than noninflamed paired samples. These results led us to consider that the production of MMPs by fibroblast-like cells may be a potential therapeutic target in diverticulitis. Traditionally, tetracycline derivates have been commonly used in the treatment of diverticulitis. Although tetracycline is an antibiotic agent, it is also an established broad-spectrum MMP inhibitor.32, 33 Thus, our results suggest that modulation of MMPs would provide an additional mechanism other than the antibacterial effect in the case of complicated diverticulitis. In the present study we also found, in general, greater percentages of expression of MMPs and lower percentages of expression of TIMPs in CD cases than in diverticulitis cases, both in inflamed samples as well as in noninflamed samples. While TIMPs inhibited the enzymatic activity of MMPs, these findings might to indicate a more controlled degradation activity of MMPs in diverticulitis compared to CD. In fact, for example, increased levels of TIMP-1 have been associated with the presence of fibrotic strictures in CD cases via inhibition of MMPs.34 On the other hand, it has been reported that MMP-3 and MMP-9 are markedly upregulated in intestinal fistulae and may contribute to fistulae formation through degradation of the extracellular matrix.35 Probably because a small number of cases showed stenosis/obstruction (n = 10) and fistulization (n = 4) in the present, study we found no significant relationship between MMPs/TIMPs expression and disease phenotype. Nevertheless, this aspect merits further investigation, including a larger patient population as well as a wider range of MMP expressions.

To differentiate diverticulitis cases, our results show higher expression of several MMPs by epithelial cells, fibroblast-like cells, or MICs from inflamed mucosas compared with noninflamed mucosas. These findings suggest crosstalk between immune and nonimmune cells in the gut, which is related with mucosal damage in CD. In fact, recently it was reported that neutrophil MMP-9 and MMP-26 and stromal TIMP-1 and TIMP-3 expressions decreased along with treatment based on immunosuppressive drugs in patients with CD.36 One of these important mediators of this crosstalk may be tumor necrosis factor (TNF). The mechanism by which TNF causes intestinal tissue injury is believed to be enhancement of MMP production at local sites.13, 37 Treatment with the anti-TNF infliximab is very effective in patients with active fistulizing CD. These observations point to an antiproteolytic and matrix protective phenotype induced by infliximab. However, despite less impressive differences in TNF secretion, MMP activity levels were much higher in intestinal bowel disease compared with controls, stressing the importance of other proinflammatory pathways in upregulating MMPs.38

In summary, our results indicate differential expression patterns of MMPs and TIMPs between CD and diverticulitis. We think that these enzymatic expressions may point to a more selective administration of MMP inhibitors as a new therapeutic strategy in these digestive disorders.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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