NDT Advance Access originally published online on June 2, 2007
Nephrology Dialysis Transplantation 2007 22(10):2838-2848; doi:10.1093/ndt/gfm323
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Inhibitory effects of matrix metalloproteinase inhibitor ONO-4817 on morphological alterations in chlorhexidine gluconate-induced peritoneal sclerosis rats
Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan
Correspondence and offprint requests to: Dr Yasuhiko Tomino, Professor, Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan. Email: yasu{at}med.juntendo.ac.jp
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Abstract |
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Top
Abstract
IntroductionBackground. The activity of gelatinase, matrix metalloproteinase-2, in effluent was increased in peritoneal dialysis patients with encapsulated peritoneal sclerosis (EPS) and in chlorhexidine gluconate-induced peritoneal sclerosing (PS) animal models. The objective of the present study was to investigate the effect of matrix metalloproteinase inhibitor (ONO-4817), an anticancer agent with anti-angiogenesis and anti-infiltration effects, on the development of peritoneal fibrosis in chlorhexidine gluconate-induced PS rats.
Methods. Forty-five Sprague–Dawley (S–D) rats were intraperitoneally injected with saline as control (n = 15) or with chlorhexidine gluconate (CH) (1.5 ml/100 g) in the CH group (n = 15). ONO-4817 (5 mg/rat) was administered intravenously to CH rats (the ONO-4817 group, n = 15) from initiation to the end of the study. After 22 days of ONO-4817 administration, the rats were sacrificed and the parietal peritoneum was harvested. The gene expressions of transforming growth factor-β (TGF-β), 
-smooth muscle actin (-SMA) and type I collagen in the peritoneum were analysed by the reverse transcription-polymerase chain reaction (RT–PCR). Peritoneal tissues were also evaluated immunohistologically.
Results. ONO-4817 significantly inhibited thickening of the submesothelial layer and accumulation of type I collagen in the peritoneum. ONO-4817 also prevented increases of the number of macrophages and blood vessels. The expressions of TGF-β, -SMA and type I collagen in the peritoneum were markedly suppressed in ONO-4817-treated rats.
Conclusion. It appears that the administration of the MMP inhibitor ONO-4817 might be a new approach to the amelioration of PS.
Keywords: angiogenesis; cell infiltration; continuous ambulatory peritoneal dialysis (CAPD); matrix metalloproteinases (MMP); sclerosing peritonitis
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Introduction |
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Peritoneal dialysis (PD) is an effective treatment for patients with end-stage renal failure (ESRF). A common functional change in the peritoneal membrane is ultrafiltration failure, which is related to duration of PD and structural alterations of the peritoneal membrane [1]. These pathological alterations of the peritoneal membrane in long-term PD patients may progress to encapsulating peritoneal sclerosis (EPS), a serious complication in PD patients. It is well known that the characteristics of peritoneal findings in long-term PD patients are severe submesothelial thickening, loss of mesothelial cells and neoangiogenesis with vasculopathy [2]. Changes of peritoneal solute transport in long-term PD patients often result from an increased vascular surface area with vasculopathy. Angiogenesis and vasculopathy in the peritoneum may play a predominant role in the regulation of water and solute transport in the peritoneum. However, the relationship between vascular changes and development of peritoneal fibrosis is still unclear.
Recently, many investigators have developed experimental models for peritoneal sclerosis (PS) in rats and mice using intraperitoneal injection of chlorhexidine gluconate (CH) [3]. Gene transfer of transforming growth factor-β (TGF-β) to the rat peritoneum revealed increased thickening of the peritoneum and large numbers of infiltrating inflammatory cells and vessels in the submesothelial compact zone [4]. We also previously reported that anti-neutralized vascular endothelial growth factor (VEGF) antibody attenuated severe interstitial fibrosis via suppression of marked angiogenesis and macrophage infiltration among serial morphologic changes in the peritoneum using the experimental PS rat model [5]. Therefore, we hypothesized that macrophage infiltration and angiogenesis in chronic inflammation may play a critical role in the development of peritoneal fibrosis.
In recent studies on peritoneal fibrosis animal models and EPS patients, an increase in MMP activity, mainly MMP-2, has been shown in peritoneal fluids and peritoneal tissues [6]. We reported a new marker for early diagnosis in PD patients with peritonitis [7]. MMP-2 is preferentially secreted by fibroblasts, epithelial cells and macrophages, while MMP-9 is preferentially released by inflammatory cells, especially neutrophils [7,8]. Matrix metalloproteinases (MMPs) play a central role in many biological processes such as embryogenesis, normal tissue wound healing and angiogenesis and in several diseases such as atheroma, arthritis, cancer and tissue ulceration that are related to the migration of cells such as cancer cells, neutrophils, macrophages, fibroblasts and endothelial cells. Recently, it was reported that matrix metalloproteinase (MMP) inhibitor prevents fibrosis in pulmonary fibrosis and myocardial infarction mouse models [9]. Hirahara et al. [6,7] suggested that MMP-2 might be associated with the progression of PS/EPS and the formation of abdominal cocoons. MMPs may play an important role in the development of peritoneal fibrosis via angiogenesis and cell infiltration. As tumour cells become free of solid tumours, pass through the vascular basement membrane consisting of type IV collagen, laminin and fibronectin and form new metastatic lesions, the tumour cells produce and release MMPs, especially gelatinase i.e. MMP-2 and MMP-9. Stimulated endothelial cells also produce MMP-2 and MMP -9 and release them in angiogenesis. Recently, many MMP inhibitors have been developed as anti-invasive agents for solid malignant tumours. Because the design of MMP inhibitors including ONO-4917 was based on the scissile site sequence of peptide substrates, with moieties customized to chelate the critical zinc ion at the active site of the enzyme, these drugs inhibit the activation of current MMPs. It has been reported that ONO-4817, an active MMP inhibitor has a broad inhibitory spectrum for MMPs and causes a significant reduction in tissue injury and inflammation through its inhibitory effect on infiltrating inflammatory cells in dextrin sulphate sodium-induced colitis in mice [10].
We demonstrated that the suppression of inflammatory cell infiltration and endothelial migration in angiogenesis by a matrix metalloproteinase inhibitor (ONO-4817) might inhibit peritoneal alteration, i.e. PS, in CH-induced PS rats.
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Materials and methods |
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Matrix metalloproteinase inhibitor
The MMP inhibitor (ONO-4817) [(2S,4S)-N-hydroxy-5-ethoxymethloxy-2-methyl-4-(4-phenoxybenzoyl) aminopentanamide, M.W. = 416.48] was developed as a new anti-angiogenic agent that inhibits the invasive properties of cancer. ONO-4817, which has a selective inhibitory spectrum, binds reversibly to the zinc-binding region of MMPs such as MMP-2, 8, 9, 12 and 13, but not MMP-1, 3 and 7 [10,11]. Angiogenesis and invasion of cancer cells were suppressed by ONO-4817 through blockade of the transformation from latent-form MMP to active-form MMP. Pharmacokinetic studies showed that plasma concentrations of ONO-4817 were >10 µmol/l at 1 h and not detected at 4 h after intravenous administration at a dose of 3 mg/kg (unpublished). This agent was kindly provided by ONO Pharmaceutical Co., Ltd., Osaka, Japan.
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Animal model |
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Forty-five male Sprague–Dawley (S–D) rats at 8 weeks of age (Charles River Breeding Laboratories, Kanagawa, Japan) were used in the present study. All animal studies were performed according to the National Research Council Guidelines. Thirty rats were given an intraperitoneal injection (ip) of 1.5 ml/100 g body weight (BW) of 0.1% CH and 15% ethanol dissolved in saline three times a week. The rats were injected with the same dosages of 15% ethanol dissolved in saline without CH as control (n = 15). These rats were sacrificed on days 0, 7, 14 and 21. Fifteen CH group rats were injected with MMP inhibitor (ONO-4817) at 5 mg via the tail vein every other day. These rats were sacrificed on days 7, 14 and 21. The animals did not show any bacterial infection in their ascites.
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Histological assessment and quantification of the submesothelial compact zone |
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The anterior abdominal walls were fixed in 10% formalin and then embedded in paraffin. The sections were stained with haematoxylin and eosin (HE) and Masson's trichrome. Both parietal and visceral peritoneal surfaces were evaluated by morphometry and immunohistochemistry. Thickening of the submesothelial compact zone, an area from the abdominal muscular surface to the peritoneal cavity, was defined as interstitial fibrosis. Quantification of the submesothelial compact zone was performed by image analysis. The images were analysed using KS400 Imaging System Release 3.0 (Carl Zeiss, Germany) as described previously [5]. Briefly, the objectives (x 200) were positioned at random on the sections. The microscopic image was recorded at each of these five positions. The thickness of the submesothelial compact zone was measured. Fibrosis was defined as a submesothelial compact zone of more than 15 µm (highest value recorded for controls).
Immunohistochemical analysis
Immunohistochemical analyses were performed using 4 µm paraffin-embedded tissue sections and frozen sections. Frozen sections were sliced with a cryostat. Paraffin-embedded tissue sections were stained using the previously reported method [5]. Briefly, the tissues were deparaffinized in xylene, followed by 100% ethanol and then placed in freshly prepared methanol/0.3% H202 solution. Microwave antigen retrieval was performed with citrate buffer. The sections were then blocked with 2% bovine serum albumin (BSA), 2% fetal calf serum (FCS) and 0.2% fish gelatin in 0.01M phosphate-buffered saline (PBS) for 30 min, followed by overnight incubation with mouse anti-human MMP-2 antibody (Daiichi Fine Chemical Co. Ltd, Toyama, Japan), mouse anti-human MMP-9 antibody (Daiichi Fine Chemical Co. Ltd, Toyama, Japan), goat anti-human VEGF antibody (Santa Cruz, CA, USA) (1:100), and goat anti-human platelet-endothelial cell adhesion molecule-1 antibody (PECAM-1, CD31) (Santa Cruz, CA, USA) (1:200) used as markers for vascular endothelial cells; mouse anti-human -smooth muscle actin (-SMA) (ARP, MA, USA), (1:50) used as a marker for myofibroblasts/fibroblasts; and mouse anti-rat ED-1 antibody (Serotec Ltd, UK), (1:1000) used as a marker for macrophages/monocytes.
Biotinylated rabbit anti-goat IgG antibody (Vector, CA, USA) (1:200) was then incubated with the avidin-biotin peroxidase complex (Vector, CA, USA) at room temperature. The bound antibodies were visualized with 3,3'-diaminobenzine containing 0.003% H2O2. Immunohistochemical staining for TGF-β (Santa Cruz, CA, USA) (1:200) was performed using the routine method with some modification. After microwave antigen retrieval, 0.25% trypsin was added for 30 min at 37°C. Type I collagen was applied to 4 µm cryostat sections with mouse anti-type I collagen antibody (LSL, Tokyo, Japan) (1:1000). Negative staining was confirmed by incubation without primary or secondary antibody. To examine the double-immunofluorescence labelling and confocal images of the parietal abdominal peritoneum, thin peritoneal sections were incubated with mouse anti-rat ED-1 antibody (fluorescein isothiocyanate: FITC) and then with goat anti-human VEGF or rabbit anti-human TGF-β antibody (rhodamine) after washing with PBS. The sections were examined using a conventional fluorescence microscope (Eclipse TE300; Nikon, Tokyo), followed by confocal laser scanning microscopy (Axiovert 100M) using the LSM 510 system (Zeiss, Tokyo). All images were obtained from individual optical sections.
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Reverse transcription–polymerase chain reaction (RT–PCR) analysis |
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Peritoneal tissue samples were snap-frozen immediately after resection and stored at –80°C for RNA extraction. Total RNA was extracted by the acid guanidinium thiocyanate-phenol-chloroform method [12]. Total RNA (1 µg) from the peritoneum was reverse-transcribed using oligo (dT) primers (Invitrogen, CA, USA) and reverse transcriptase (Superscript II; Invitrogen, CA, USA). First-strand cDNA was synthesized by priming 1 µg of total RNA in a 20 µl RT mixture containing 4 µl of 5 x first-strand buffers, 1 µl of deoxynucleotide triphosphate (dNTP) (Invitrogen) mix containing 10 mmol/l of each dNTP base, 2 µl of 0.1 mol/l dithiothreitol (DTT), and 200U of reverse transcriptase. After incubation at 42°C for 50 min, the reaction was stopped by heating at 90°C for 5 min. To remove RNA complementary to the cDNA, 1 µl of RNase H (Invitrogen) was added to the product and incubated at 37°C for 20 min. The product was diluted 10 times and used for PCR. Then 5 µl of the RT product was added to the reaction mixture containing 2 µl of PCR buffer diluted 10 times, 1.6 µl of 2.5 mmol/l dNTP mixture, 1 U of Taq polymerase (Takara Biochemicals, Ohtsu, Japan) and 2.5 pmol/µl of each primer in a final volume of 20 µl.The regions amplified by each set of primers are shown in Table 1. The mixture was denatured and amplified in the Gene Amp PCR System Peltier Thermal Cycler-200 (MJ-JAPAN, Tokyo, Japan) under the following conditions: 1 min at 94°C, 1 min at 57 to 62°C and 1 min at 72°C for 22 to 35 cycles, (annealing temperatures: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 57°C; MMP-2, 54°C; TIMP-2, 54°C;
-SMA,58°C; VEGF,60°C; TGF-β,60°C and type I collagen, 59°C; cycles: GAPDH, 22; MMP-2,27; TIMP-2,27; -SMA,22; VEGF,33; TGF-β, 30; and type I collagen 1, 23). Negative controls (cDNA-free solutions) were included in each reaction. Quantification of the PCR products of all samples was performed three times.
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After defined cycles of PCR, 5 µl of the 20 µl reaction volume was electrophoresed on 1.5% agarose gel in 1 x Tris-acetate EDTA and amplified bands were detected by ethidium bromide staining. The intensity of ethidium bromide fluorescence was obtained using BIO-PRINT (Vilber Lourmat, FRANCE) and measured using Master Scan (Scanalytics, MA, USA) as described previously [4]. Initially, each cDNA species was amplified between 20 and 40 cycles. The optimum number of PCR cycles for each cDNA species was determined by plotting the PCR product yield of different cycles on a semi-logarithmic graph and the cycle number representing the exponential amplification was chosen for the final amplification. For quantification of the PCR products of all samples, the samples were evaluated in comparison with the PCR product to GAPDH. The fidelity of the PCR products was also confirmed by nucleotide sequencing. Negative controls (cDNA-free solutions) were included in each reaction. The regions amplified by each set of primers were chosen from published documents as follows: rat VEGF 310 bp [5], rat TGF-β 441 bp [13], rat
-SMA 389bp [14], rat type I collagen 296 bp [15] and GAPDH 707 bp [5]. |
Quantitative analysis for blood vessel and macrophage assessment |
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Peritoneal tissues from the anterior abdominal walls were stained with goat anti-human PECAM-1 (CD31) antibody as a marker for vessels and ED-1 antibody as a marker for macrophages/monocytes by immunohistochemistry. Quantification of the submesothelial compact zone, i.e. the areas between mesothelium and interstitial smooth muscle layer, was performed by image analysis. The images were analysed using a KS400 imaging System Release 3.0 (Carl Zeiss, Germany). The numbers of blood vessels and infiltrating macrophages and monocyte were evaluated in all segments of the section. To avoid selection bias, quantification was performed in a pre-defined segment. The objective (x 200) was positioned and the microscopic image was recorded at each of the five positions except artefacts. The numbers of vessels and macrophages in each field were measured and their averages were calculated.
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Statistical analysis |
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The data are presented as mean ± SD. ANOVA and the unpaired t-test were used to test statistical significance. The level of significance was set at P < 0.05.
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Results |
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Effect of chlorhexidine gluconate (CH) on the structure of the peritoneum
No marked change was observed in peritoneum in the control rats at days 7, 14 and 21 after saline treatment (Figure 1A, B and C). CH induced thickening of the submesothelial compact zone was gradually increased (Figure 1D, E, F). There was a non-specific inflammatory response with edematous thickening and cellularity at day 7 (Figure 1D). Thickening and cellularity were exaggerated with vascularization with increases in collagen tissues at days 14 and 21 (Figure 1E and F). At days 14 and 21 in the ONO-4817 group (Figure 1H and I), thickening of the compact zone, cellularity and vascularization were significantly suppressed compared with those in the CH group (Figure 1E and F). In the ONO-4817 group, thickening of the submesothelial compact zone was markedly suppressed compared with that in the CH group at days 14 and 21 (P < 0.01, Figure 1H, I). There were marked differences in the thickness of the peritoneum in each observation between the CH group and the ONO-4817 group. The thickness of the peritoneum at day 21 was 9.64 ± 1.92 µm in the control group, 388.6 ± 91.0 µm in the CH group and 142.7 ± 14.2 µm in the ONO-4817 group (Figure 2).
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Immunohistochemical analysis of peritoneal fibrosis |
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The appearance of peritoneal fibroblasts/myofibroblasts and accumulated collagen in the peritoneal tissues is shown in Figure 3. Some of the cells infiltrating the submesothelial areas such as fibroblasts/myofibroblasts showed positive
-SMA staining in the CH and ONO-4817 groups at day 21 (Figure 3B and C). Accumulation of type I collagen in the submesothelial compact zone was increased at day 21 in the CH and ONO-4817 groups (Figure 3E and F). The expressions of -SMA and type I collagen in the ONO-4817 group were markedly decreased compared with those in the CH group (Figure 3C and F). No histological change was observed in the control group (Figure 3A and D).
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Some of the interstitial cells and endothelial cells in the submesothelial compact zone expressed MMP-2 in the CH and the ONO-4817 groups (Figure 4A). some of the interstitial, but none of the endothelial cells, showed MMP-9 positive in those groups (Figure 4B). There was no significant difference of MMP expression between the CH group and the ONO-4817 group (data not shown).
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Gene expression in the mRNA assessment |
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RNA was isolated from parietal peritoneal tissues at day 21 and analysed by RT-PCR. Expressions of MMP-2, TIMP-2, VEGF,
-SMA, TGF-β and type I collagen in the parietal peritoneum were enhanced in the CH group (Figure 5). In the ONO-4817 group, the levels of VEGF, TGF-β and type I collagen mRNA expressions after intraperitoneal injection of CH were significantly suppressed compared with those in the CH group at day 21 (P < 0.001) (Figure 5) [3,5,6]. MMP-2 and -SMA mRNA expression in the ONO-4817 group also showed a statistically significant decrease compared with those in the CH group (P < 0.05) (Figure 5) [1,4].
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Immunohistochemical and quantitative analyses for blood vessel and macrophage assessment |
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Immunohistochemical analyses demonstrated the appearance of CD31-positive interstitial cells as a marker of vascular endothelial cell in both the CH and ONO-4817 groups. CD31-positive cells were strongly expressed in the deep part of the submesothelial area (Figure 6A). CH significantly stimulated angiogenesis in the peritoneum in both the CH and ONO-4817 groups (P < 0.001, respectively) (Figures 6B, 4A). The increase of blood vessels in the submesothelial compact zone in the CH group was significantly suppressed by ONO-4817 administration (P < 0.01) (Figure 6B). ED-1 positive cells as a marker for infiltrating cells with staining of monocytes and macrophages were localized in the submesothelial compact zone at day 21. The numbers of ED-1 positive cells were increased at day 21 in both the CH and ONO-4817 groups (Figure 6C). The increase of ED-1 positive cells in the submesothelial compact zone in the CH group was significantly suppressed by ONO-4817 administration (P < 0.01) (Figures 6D, 4B).
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VEGF-positive cells were spread diffusely over the submesothelial area (Figure 7A and B). Most infiltrated macrophages (ED-1 positive cells, in red) in the submesothelial compact zone demonstrated expression of VEGF (in orange) (Figure 7C and D). TGF-β positive cells also showed diffuse distribution in the submesothelial compact zone (Figure 7E and F). TGF-β (in green) was demonstrated in ED-1 positive cells and other interstitial cells (Figure 7G). Some ED-1 positive cells were positive for TGF-β (in orange) (Figure 7G and H).
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Discussion |
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Pathological changes in the peritoneal membrane with long-term PD are characterized by a decrease or loss of mesothelial cells, enlargement of the submesothelial compact zone due to interstitial fibrosis accompanied by degeneration of collagens and vasculopathy. We clarified the influence of angiogenesis on peritoneal thickening and fibrosis using matrix metalloproteinase (MMP) inhibitor ONO-4817, which inhibits metastasis, invasion of malignant cells and angiogenesis. In this study, thickening of the peritoneum was markedly suppressed via a decrease in the number of macrophages and blood vessels in the submesothelial compact zone with simultaneous administration of ONO-4817 in PS rats. Macrophages infiltrating in the submesothelial compact zone expressed TGF-β1 and VEGF. Both capillary endothelial cells and myofibroblasts also expressed TGF-β1. Expressions of VEGF, TGF-β,
-SMA and type I collagen mRNA in the peritoneum were suppressed by ONO-4817 treatment. ONO-4817 significantly inhibited thickening of the submesothelial layer and expression of -SMA and type I collagen in the peritoneum. Our approach for rescue from peritoneal fibrosis using an MMP inhibitor was associated with breaking the vicious cycle of monocyte infiltration, collagen deposition and neoangiogenesis. There are several factors implicated in the development of PS in PD patients. The most important factor is the conventional bioincompatible PD solution, which contains a high concentration of glucose and glucose degradation products (GDPs), such as methylglyoxal, glyoxal or 3-deoxyglucosone, which are produced in peritoneal dialysate during the process of heat sterilization. Advance Glycation Endproducts (AGEs) are also formed in the peritoneal cavity during peritoneal dialysis. Uraemia is considered as a cause of PS, since even before initiation of dialysis, ESRF patients have a thickened peritoneum when compared with the peritoneum in healthy control subjects [2]. Inflammatory cytokines, which are induced in the peritoneal cavity during peritonitis, may further promote chronic inflammation and fibrosis. These stimuli enhance the production of fibrogenic and angiogenic mediators in the mesothelial cells, infiltrating cells and vascular cells. CH is not used clinically in PD and is a non-specific inducer of peritoneal fibrosis in experimental PS in studies of pathogenesis. However, the PS rat model with repeated intraperitoneal injections of CH should be appropriate for our purpose of clarifying the influence of cellular infiltration and marked neoangiogenesis on the development of interstitial fibrosis and thickening of the interstitial zone. Recently, it was reported that MMPs, especially gelatinases (MMP-2 and MMP-9), are involved in pulmonary fibrosis, liver fibrosis and myocardial infarction in mouse models [9]. MMP-2 and MT-1 MMP mRNA in pulmonary fibroblasts and macrophages were over-expressed in pulmonary fibrosis animal models [9]. We previously reported that macrophage-derived MT-1 MMP and increased MMP-2 activity are associated with glomerular damage in crescentic glomerulonephritis [14]. MMP-2 might be associated with the progression of PS/EPS and the formation of an abdominal cocoon in the PS rat [6]. Therefore, we hypothesized that simple sclerosis may be an indispensable prerequisite for development of EPS and MMPs. MMP inhibitors, developed as prevention of tumour metastasis and neoangiongenesis [11], also suppress inflammation through inhibition of the migration of infiltrating inflammatory cells [10]. Protease inhibitors are able to inhibit invasion but not proliferation via production of endogenous and exogenous TGF-β1 in pancreatic cancer cells [16]. Serial morphological changes in the PS rats were characterized by marked cellular infiltration and interstitial oedema prior to loss of mesothelial cells, prominent neoangiogenesis and eventual severe thickening of interstitial fibrosis [5]. Macrophages accumulate in the peritoneum and are activated by stimulated resident cells, including mesothelial cells, fibroblasts and infiltrating macrophages at an early stage of PS. The infiltrating macrophages are able to accelerate peritoneal fibrosis by stimulating fibroblast growth factor production from these peritoneal cells as mentioned in previous reports [17]. Since inflammatory cells or tumour cells release MMPs that degrade extracellular matrix during migration, the MMP inhibitor ONO-4817 could also prevent inflammatory cells from slipping through the basement membrane and intercellular spaces of peripheral vessels. The significant reduction in the number of macrophages in the ONO-4817 group suggested an inhibitory effect on inflammatory cell recruitment. This hypothesis is based on previous studies about the role of MMPs in cell migration [18]. Neoangiogenesis, especially VEGF production by mesothelial cells, also prompts fibrosis. Angiogenesis is a process by which new blood vessels are formed from pre-existing vessels. New blood vessel formation by angiogenesis involves the degradation of extra-cellular matrix combined with sprouting and migration of endothelial cells from pre-existing capillaries. Thus, endothelial cells release MMPs by themselves for the proliferation, migration and differentiation as well as the degradation and subsequent re-establishment of the basement membrane. Increase of CD31-positive cell numbers was suppressed by ONO-4817 treatment in this study. The expression of VEGF mRNA was also markedly inhibited in the ONO-4817 group. Therefore, ONO-4817 inhibited neoangiogenesis directly by blockade of MMP activity and indirectly by suppression of angiogenic growth factor, i.e. VEGF production.
Since VEGF blockade by the neutralizing antibody revealed significant suppression of neoangiogenesis and submesothelial thickening in our previous study [5], VEGF may also elicit peritoneal fibrosis via neoangiogenesis of submesothelial tissues as an angiogenic growth factor. Several investigators reported that macrophages and fibroblasts producing VEGF provide a basic framework for proliferating endothelial cells that form vascular structures. TGF-β has been shown to be a potent promoter of collagen gene expression, leading to enhanced production of type I and type III collagens. The direct relationship between VEGF and TGF-β in the development of peritoneal fibrosis is still unclear. Therefore, it was suggested that marked angiogenesis promoted recruitment of the VEGF and TGF-β producing cells, which were able to alter tissue structure and initiate a vicious cycle in peritoneal fibrosis.
The levels of MMP-2 in peritoneal effluent and tissues were mainly up-regulated in CH animal models as well as in PD patients with EPS [6]. ONO-4817 suppresses the activation of a wide range of MMPs such as collagenase, stromelysins and gelatinases (MMP-2 and MMP-9). Amelioration of inflammatory injury through inhibition of activating MMPs contributes to the initiation/amplification of tissue injury in dextrin sulphate sodium-induced colitis in response to ONO-4817 as observed on day 3 [19]. Based on the pharmacokinetics of ONO-4817 [20], a dose of 5 mg, i.v. every other day was selected for our study and morphological findings were evaluated on days 7, 14 and 21.
In the present study, blockade of infiltration of inflammatory cells and angiogenesis using MMP inhibitor induced marked suppression of peritoneal fibrosis in the CH rat model. This study provides the first evidence that ONO-4817, a synthetic MMP inhibitor, may have therapeutic potential in the treatment of peritoneal fibrosis and suggests that administration of MMP inhibitor ONO-4817 might be a new approach to amelioration of excessive PS in PD patients.
Conflict of interest statement. None declared.
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Accepted in revised form: 2. 5.07
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