Clarithromycin Attenuates Acute and Chronic Rejection Via Matrix Metalloproteinase Suppression in Murine Cardiac Transplantation
Masahito Ogawa, BS*,
Jun-ichi Suzuki, MD*,*,
Keiichi Hishikari, BS*,
Kiyoshi Takayama, PhD*,
Hiroyuki Tanaka, MD, PhD and
Mitsuaki Isobe, MD, PhD*
* Department of Cardiovascular Medicine, Tokyo Medical and Dental University, Tokyo, Japan
Department of Thoracic Surgery, Tokyo Medical and Dental University, Tokyo, Japan.
Manuscript received October 17, 2007;
revised manuscript received December 13, 2007,
accepted January 21, 2008.
* Reprint requests and correspondence: Dr. Jun-ichi Suzuki, Department of Cardiovascular Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 Japan. (Email: jsuzuki.cvm{at}tmd.ac.jp).
 |
Abstract
|
|---|
Objectives: Clarithromycin (CAM), a major macrolide antibiotic, has many biological functions, including matrix metalloproteinases (MMPs) regulation. However, little is known about the effect of CAM in heart transplantation via MMP-9. The purpose of this study was to clarify the role of MMPs regulated by CAM in the progression of rejection.
Background: The MMPs are critical in the development of inflammation and tissue remodeling. The MMP-9 level is associated with the rejection of heart transplantation.
Methods: We orally administered CAM into murine cardiac allograft recipients. Total allomismatch combination and class II mismatch combination were used for the analysis of graft survival, pathology and molecular.
Results: Clarithromycin improved acute rejection judged by graft survival and by myocardial cell infiltrating area in a total allomismatch combination. The CAM-treated allografts showed affected expression of T-cells, macrophages, and MMP-9 in immunohistochemistry. Zymography indicated that enhanced MMPs activities were observed in nontreated hearts, whereas CAM suppressed the levels. In chronic rejection, CAM suppressed the development of graft arterial disease and myocardial remodeling compared with that of nontreatment. Clarithromycin inhibited the expression of MMP-9, whereas the treatment did not alter the expression of MMP-2 and tissue inhibitor metalloproteinase-1 in macrophages and smooth muscle cells. Inhibition of MMP-9 by CAM was associated with suppression of smooth muscle cell migration and proliferation.
Conclusions: Clarithromycin is useful to suppress allograft remodeling, because it is critically involved in the prevention of cardiac rejection through the suppression of MMP-9.
|
Abbreviations and Acronyms
| | CAM = clarithromycin | | GAD = graft arterial disease | | DMEM = Dulbecco's modified Eagle's medium | | ICAM = intercellular adhesion molecule | | IL = interleukin | | MHC = major histocompatibility complex | | MMP = matrix metalloproteinase | | mRNA = messenger ribonucleic acid | NF- B = nuclear factor-kappa B | | RNA = ribonucleic acid | | SMC = smooth muscle cell | | TIMP = tissue inhibitor metalloproteinase |
|
Acute cardiac rejection is still a major complication of heart transplantation. Inflammatory factors such as cytokines, chemokines, and adhesion molecules play a critical role in the development of acute rejection (1–3). The allografts show diffuse arterial neointimal formation (graft arterial disease [GAD]), that consists of smooth muscle cells (SMCs), extracellular matrix (ECM), and variously mononuclear leukocytes during long-term observation. Although GAD ultimately culminates in vascular stenosis and ischemic graft failure, the infallible care and prevention measures are still unknown.
Clarithromycin (CAM) is known as a 14-member ring macrolide and a potent antibiotic for the treatment of various microbial infections. Recently, CAM has been reported to have multiple biologic effects, such as alteration of inflammatory factors (4,5). Many inflammatory cells, such as lymphocytes (6,7) and monocytes/macrophages (8,9), produce matrix metalloproteinases (MMPs), and levels have been shown to be upregulated in grafts with human and various mammal organ transplantation (10–12). The MMPs are a large family of proteinases that proteolytically degrade ECM such as collagen and proteoglycan. The degradation of ECM is an important event in the process of inflammation and tissue remodeling. A member of MMPs, MMP-9 (gelatinase B) is known to play an important role in tissue remodeling and the migration of various cells, such as SMCs (13,14), macrophages, and other cells. Smooth muscle cells are shown to express MMP-2 and -9, and excess activation of MMP-2 and -9 induce the destruction of the ECM and can lead to pathological remodeling and vascular restenosis. However, little is known about the role of MMP-9 for acute and chronic rejection in heart transplantation. This study demonstrated that CAM suppressed acute and chronic rejection through the inhibition of the MMP-9 expression from mononuclear cells and SMCs.
|
Methods
|
|---|
Reagents.
The CAM was kindly provided by Taisho Toyama Pharmaceutical Corporation, Ltd (Tokyo, Japan).
Experimental animals and cardiac transplantation.
The male BALB/c (H-2d) and C57BL/6 (H-2b) mice (4 to 6 weeks, 20 to 25 g) were obtained from Japan Clea Corporation (Tokyo, Japan), and this combination was used as the major histocompatibility complex (MHC) total allomismatch group for analysis of acute rejection (8 animals for graft survival analysis in each group). A combination of male C57BL/6 Bm12 (H-2bm12) and C57BL/6 mice (4 to 6 weeks, 20 to 25 g) was used as the MHC class II mismatch group for analysis of chronic rejection (n = 6 each group). Heterotopic cardiac transplantation was performed as described previously (15). Recipient mice were given oral administration CAM (100 mg/kg/day) twice/day. We selected the dose for mice on the basis of previous papers (16,17). Graft survival and function were evaluated by daily palpation, and cessation of beating was interpreted as rejection as previously reported (18). The mortality rate induced by surgical or technical failure of this murine heart transplant procedure is <2% in our laboratory. This investigation conforms with the Guide for the Care and Use of Laboratory Animals in the Tokyo Medical and Dental University.
Histopathology.
Histopathological analysis was performed as described previously (19). We obtained 5 transverse sections/heart for histopathologic examination. The sections were stained with hematoxylin and eosin, Elastica van Gieson, and Mallory. The area of myocardium and surrounding tissue affected by acute rejection and chronic rejection were determined with a computer-assisted analyzer (Image-Pro Express, Nikon, Tokyo, Japan). The area ratio (affected/entire area as a percentage) was calculated as described previously (15). All data were analyzed in a blind fashion by 2 independent investigators and averaged. Samples from 9 animals in each group of the total allomismatch combination and 6 animals in each group of the MHC class II mismatch combination were used for histological analysis.
Film in situ zymography.
Film in situ zymography was performed as described previously (20). Frozen sections were cut to a thickness of 7 µm (4 samples in each group). These sections were mounted on polyethylenetelephthalate film coated with gelatin (FIZ-GN) (Wako Pure Chemical Industries, Ltd., Osaka, Japan). As a control, these sections were mounted on polyethylenetelephthalate film coated with gelatin and an MMP inhibitor (FIZ-GI) (Wako). Frozen sections on these films were incubated in a moist chamber at 37°C for 6 h. After incubation, these films dried for 30 min and were then stained with Biebrich Scarlet Stain Solution (Wako) for 4 min. Gelatinolytic activity is visible as a clear area unstained by Biebrich Scarlet; this solution stains only gelatin on the film, leaving areas with gelatinolytic activity unstained and bright.
Immunohistochemistry.
Sections (4 samples in each group) were incubated with primary antibodies against murine intercellular adhesion molecule (ICAM)-1 (YN1/1.7) (provided by Professor Ko Okumura, Juntendo University), CD4, CD8, CA11b (BD Biosciences Pharmingen, San Diego, California), nuclear factor-kappa B (NF-B) p65, or MMP-9 (Santa Cruz Biotechnology, Inc., Santa Cruz, California) at 4°C for 12 h. Antibody-HRP conjugate was detected with Histofine Simplestain Kit (Nichirei Corporation, Tokyo, Japan), used according to the manufacturer's instructions. Enzyme activity was detected with 3-Amino-9-ethylcarbazole.
Ribonuclease protection assay.
Trizol (Invitrogen Corporation, Carlsbad, California) was used to isolate total ribonucleic acid (RNA) according to the manufacturer's protocol. The probe was synthesized by the in vitro transcription method with a Multi-Probe Template Set mCK-1 (Pharmingen), T7 polymerase, and [32P] UTP. Messenger ribonucleic acid (mRNA) levels were quantified and normalized against levels of glyceraldehyde-3-phosphate dehydrogenases (GAPDH) (4 samples in each group).
Cell preparations.
Primary SMCs were obtained from the thoracic aortas of Bm12 mice by the explant technique described previously (19). The SMCs were grown with Dulbecco's modified Eagle's medium (DMEM) (Sigma Chemical Corporation, St. Louis, Missouri) supplemented 50 µg/ml streptomycin, 50 IU/ml penicillin, and 10% fetal bovine serum at 37°C and 5% carbon dioxide. The J774.1A (BALB/cA) cells were obtained from Riken Bioresource Center (Tsukuba, Japan) and were grown with RPMI1640 (Sigma) supplemented 10% fetal bovine serum.
Western blotting.
Western blot analysis was described previously (21). The polyvinylidene difluoride membrane incubated with primary antibody MMP-9, MMP-2, and beta-actin (Santa Cruz). And then, the membrane enhanced chemiluminescence reagent (Pierce Biotechnology Inc., Rockford, Illinois). Enhanced chemiluminescence was detected with an LAS-1000 (Fujifilm Corporation, Tokyo, Japan). The data were obtained from 3 independent experiments (4 samples in each group).
Reverse transcriptase-polymerase chain reaction.
Serum-starved-J774.1A cells were stimulated with interleukin (IL)-1beta recombinant (10 ng/ml) (R&D Systems, Minneapolis, Minnesota). Serum-starved SMCs were stimulated with PDGF-BB (5 ng/ml) (Biosource International, Camarillo, California). Total RNA were collected 16 h after stimulation and isolated according to the manufacture's protocol by Trizol (Invitrogen). Complementary deoxyribonucleic acid was prepared with the reverse transcriptase-polymerase chain reaction (RT-PCR) kit. The PCR was performed with PCR-kit in the presence of oligo-primers for MMP-9, MMP-2, tissue inhibitor metalloproteinase (TIMP)-1, and GAPDH. The data were obtained from 3 independent experiments (4 samples in each group) (Table 1).
Transmigration assay.
The migration assay was performed as described previously (9,22). Briefly, the assay was performed with Transwell chambers (Corning Corporation, Corning, New York) 24-well tissue culture plates composed of 5 µm pore polycarbonate filters. The upper chambers were not coated. The SMCs were seeded at 1 x 104 cells/well in 100 µl medium. The lower chamber was filled with 400 µl of DMEM medium supplemented with 5 ng/ml PDGF-BB and 0.5% fetal bovine serum (FBS), and then the chambers were incubated at 37°C for 24 h in 5% carbon dioxide incubator. Cells were counted under a microscope, and the cells that migrated to the lower chamber were examined. The data were obtained from 3 independent experiments (4 samples in each group).
Proliferation assay.
The SMCs were seeded on the 96-well plates (5 x 103 cells/well) and then incubated with DMEM supplemented 0.5% FBS and PDGF-BB for 2 days in incubator. Cell proliferation was then assessed with the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions. Cell proliferation was expressed as the optical density (15). The data were obtained from 3 independent experiments (6 samples in each group).
Statistical analysis.
All data are expressed as mean ± SEM. Kaplan-Meier analysis was used to estimate graft survival, and the Mann-Whitney U test was used for survival differences between the 2 groups. Data were compared, and differences between the 2 groups were analyzed by the Student t test for histopathological analysis and ribonuclease protection assay. One-way analysis of variance was used in the SMC proliferation assay and SMC migration assay. Differences with values of p < 0.05 were considered significant.
|
Results
|
|---|
Graft survival prolonged and affected the histopathology by CAM.
In the major mismatch group, nontreated allografts were acutely rejected (7.3 ± 0.2 days, n = 8). However, CAM administration significantly prolonged allograft survival (9.7 ± 0.2 days, p < 0.05, n = 8) in this model (Fig. 1). Moderate myocardial cell infiltration was observed in nontreated allografts on day 7 (35.7 ± 1.9%, n = 9), whereas CAM treatment markedly attenuated myocardial cell infiltration (19.7 ± 3.0%, p < 0.05, n = 9) (Figs. 2A and 2B). Although significant fibrosis was observed in the nontreated allografts (39.6 ± 1.4%, n = 9), CAM treatment attenuated fibrosis area (23.4 ± 3.4%, p < 0.05, n = 9) (Figs. 2A and 2B).
View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 1 Survival of Cardiac Allografts
Representative data of graft survival in the full allomismatch combination. Mice treated with clarithromycin (CAM) (open circles) showed prolonged cardiac allograft survival in comparison with the nontreated mice (open squares). Nontreated allografts were acutely rejected (7.1 ± 0.2 days, n = 8). However, CAM administration statistically prolonged allograft survival (9.7 ± 0.2 days, n = 8). *p < 0.05 versus nontreated group.
|
|
View larger version (58K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 2 Pathological Findings
Representative light micrographs of allografts from the nontreated group and the clarithromycin (CAM)-treated group. (A) Representative pathological findings with hematoxylin and eosin (HE) (a, b, e, and f) and Mallory (c, d, g, and h) stain in full allomismatch allografts. Although myocardial cell infiltration and fibrosis were observed in nontreatment (a, c, e, and g) on day 7, the CAM treatment (b, d, f, and h) markedly attenuated them. Scale bars = 2 mm (a to d) and 50 µm (e to h). (B) Representative pathological findings with HE (a and b), Mallory (c and d), and Elastica van Gieson (EvG) (e and f) stain in class II mismatch allografts. Although myocardial cell infiltration, fibrosis, and graft arterial disease (GAD) were observed in the nontreated allografts (a, c, and e) on day 60, CAM treatment (b, d, and f) markedly attenuated them. Scale bars = 2 mm (a to d) and 25 µm (e and f). (C) Quantitative data of cell infiltration (a) and fibrosis (b) in the full allomismatch combination. Quantitative data of cell infiltration (d), fibrosis (e) area, and percentage of neointimal thickening (c) in the class II mismatch combination. *p < 0.05 versus nontreated group.
|
|
In the class II mismatch group, all cardiac allografts kept beating during the observation period and graft function was not different between the groups. Pathologically, severe myocardial cell infiltration (33.3 ± 1.6%, n = 6) and fibrosis (41.8 ± 0.9%, n = 6) were observed in the nontreated group, whereas CAM treatment significantly suppressed infiltration (15.9 ± 4.1%, n = 6, p < 0.05 vs. nontreated group) and fibrosis (20.0 ± 5.5%, n = 6, p < 0.05 vs. nontreated group) (Figs. 2C and 2D). The heavy neointimal thickening was observed in the coronary arteries of untreated allografts in this combination. However, intimal thickening was attenuated in the CAM-treated group (Fig. 2C [c]).
Inhibition of inflammatory factors.
Immunohistochemically, enhancement of CD4, CD8, CD11b, NF-B p65, and ICAM-1 expression was observed in the nontreated allografts of total allomismatch combination. However, CAM treatment markedly attenuated expression of these factors (Fig. 3).
View larger version (112K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 3 Immunohistochemistry
Representative immunohistochemistry of allograft from the nontreated group and the clarithromycin (CAM)-treated group. The sections were incubated with CD4 (a and b), CD8 (c and d), CD11b (e and f), intercellular adhesion molecule (ICAM)-1 (g and h), and nuclear factor-B (NF-B) p65 (i and j). Although CD4+
(a), CD8+
(c), and CD11b+
(e) cells were observed in nontreatment on day 7, CAM treatment markedly attenuated them (b, d, and f). The expression of ICAM-1 (g) and NF-B p65 (i) were observed in nontreatment myocardial infiltrating area; however, CAM treatment markedly attenuated them (h and j). Scale bars = 50 µm (a to j).
|
|
Ribonuclease protection assay was used to examine expression of cytokine mRNA from full allomismatch cardiac grafts on day 7. The mRNA levels of interferon-gamma, IL-6, IL-10, and IL-15 were significantly suppressed in the CAM-treated group compared with those of the nontreated group (Figs. 4A and 4B) (n = 4 each).
View larger version (17K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 4 RPA
(A) Representative gels in ribonuclease protection assay (RPA). Messenger ribonucleic acid (mRNA) was isolated from cardiac allografts on day 7. (B) The quantitative results. Increase of interleukin (IL)-10, IL-15, IL-6, and interferon (INF)-gamma mRNA levels were observed. However, clarithromycin (CAM) treatment markedly attenuated them. *p < 0.05 versus nontreated group. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
|
|
In vivo MMP inhibition.
The MMP activity was markedly enhanced in the infiltrated area in the nontreated full allomismatch group; however, CAM-treated grafts attenuated the MMP activity (Fig. 5A). The MMP inhibitor (+) films show the non–gelatinase-specific activity.
View larger version (98K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 5 MMP Activity
(A) Representative results of gelatinase's activity. In the matrix metalloproteinases (MMPs) inhibitor (–) group, gelatinase's activity was generally observed in the myocardial infiltrating area in full allografts (a). However, clarithromycin (CAM) treatment markedly attenuated gelatinase's activity (b). The MMPs inhibitor (+) films show the non–gelatinase-specific activity (c and d). Scale bars = 4 mm (a to d). (B) Representative results of immunohistochemistry. Although expression of MMP-9 was observed in the infiltrating cells in nontreatment allografts (a), CAM treatment suppressed the expression (b). Scale bars = 50 µm (a and b).
|
|
Immunohistochemically, although the nontreated group enhanced the MMP-9 expression in the infiltrating cells, CAM suppressed the expression in total allomismatch combination (Fig. 5B).
In vitro MMP inhibition.
Serum-starved-J774.1A cells were stimulated with 10 µg/ml IL-1beta recombinant, and after 24 h of stimulation the protein was collected. Although the control group (supplied IL-1beta and dimethyl sulfoxide as a vehicle) markedly enhanced the expression of MMP-9 compared with the native group (nonstimulated), CAM-treated cells significantly suppressed the expression of protein levels. The MMP-2 levels were comparable between the control group and the CAM-treated group (Fig. 6A [a]). The CAM-treated group altered the expression of MMP-9 mRNA but not MMP-2 and TIMP-1 mRNA (Fig. 6A [b]).
View larger version (68K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 6 In Vitro MMP-9 Inhibition
(A) Representative Western blot data detecting the expression of matrix metalloproteinases (MMP)-9 and -2 proteins in macrophages (a). The control group (Co) markedly enhanced the expression of MMP-9 compared with native (Na). The 0.2- and 20-µmol/l clarithromycin (CAM) treatment (0.2 CA and 20 CA) altered the MMP-9 protein levels compared with the control group. However, MMP-2 expression was not altered by CAM treatment. (b) Representative gels showed the expression of MMP-9, MMP-2, and tissue inhibitor metalloproteinase (TIMP)-1 messenger ribonucleic acid (mRNA) levels by reverse transcriptase-polymerase chain reaction (RT-PCR). The CAM treatment altered the expression of MMP-9 but not MMP-2 and TIMP-1. The treatment of 20 µmol/l CAM inhibited expression of MMP-9 more effectively than that of 0.2 µmol/l. (B) Representative RT-PCR data detecting the expression of MMP-9, MMP-2, and TIMP-1 mRNA in the smooth muscle cells (SMCs). The control group markedly expressed MMP-9, and CAM treatment inhibited the expression of MMP-9 mRNA levels. The CAM treatment did not alter the MMP-2 and TIMP-1 expression. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
|
|
Also, SMCs showed that MMP-9 mRNA level was markedly enhanced in the nontreated cells, whereas CAM treatment suppressed the expression of MMP-9 mRNA (Fig. 6B). However, CAM treatment did not alter the mRNA levels of MMP-2 and TIMP-1 compared with the control group.
Migration and proliferation of SMCs.
In proliferation assay of SMCs, PDGF-BB–stimulated SMCs were significantly proliferated, whereas the CAM treatment (2.0 and 20 µmol/l) attenuated the cell proliferation (p < 0.05 vs. control group) (Fig. 7A). The migration assay also showed that enhanced migration into the lower wells was observed in the control group, whereas the CAM-treated group inhibited the migration (p < 0.05 vs. control group) (Figs. 7B and 7C).
View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
|
Figure 7 Proliferation and Migration Assays
(A) Quantitative data of proliferation assay. Markedly increase proliferation of the smooth muscle cells (SMCs) was observed by stimulation of PDGF-BB (Biosource International, Camarillo, California) compared with those of the native group. The 0.2-µmol/l clarithromycin (CAM) treatment did not significantly inhibit the cell proliferation, whereas 2.0- and 20-µmol/l CAM treatment significantly inhibited the proliferation compared with the control group. *p < 0.05 versus control group. (B) Representative light micrographs of migration assay. Although the SMCs markedly migrated to a lower well by the stimulation of PDGF-BB (b) compared with those in the native group (a), CAM (20 µmol/l) treatment (c) significantly suppressed the SMC migration. (C) The quantitative data of migration assay. Counts of SMCs in a lower well significantly decreased in CAM-treatment group compared with the control group. *p < 0.05 versus control group.
|
|
|
Discussion
|
|---|
Clarithromycin is not only a potent antibiotic for the treatment of various microbial infections but also has multiple biologic effects. Although the 16-member ring macrolide do not have multiple effects, it is believed that the multiple effects by a macrolide might be characteristic of 14-member ring macrolides (9,23). In this report, we have newly demonstrated that CAM inhibited expression of MMP-9 and suppressed acute and chronic rejection in murine transplantation models.
Firstly, we demonstrated that CAM suppressed the acute rejection in the MHC total allomismatch combination group. We showed that CAM treatment suppressed MMP-9 and NF-B activity in this allograft model with zymography and immunohistochemistry. The T lymphocytes and macrophages are a major component of the cellular infiltration in allografts rejection (24,25); they contribute to the development of tissue injury in acute inflammatory reaction (25,26). The MMPs play an important role in cell migration (27) and are involved in the migratory capabilities of inflammatory cells such as T-cells (6). The MMP-9 is known to be especially important in inflammation, and the gelatinase have been shown to play a critical role in the process of the infiltration in inflamed tissues (28). We demonstrated that CAM attenuated CD4+, CD8+, and CD11b+ cell infiltration, altered the expression of inflammatory cytokines, and consequently decreased the infiltration and fibrosis compared with the control group in cardiac allografts. It has previously been reported that CAM inhibits the activation of NF-B and activator protein-1 (4,5). The interaction between ICAM-1 and the receptor lymphocyte function-associated antigen-1 affects both alloantigen specific and nonspecific phases of graft destruction after heart transplantation. The ICAM-1 is regulated by NF-B, which is a key transcription factor of inflammation or immunosuppression (29). At this point, there has been no report to compare the immunological effects in transplantation between CAM and immunosuppressive agents directly in vivo. However, there was a report to clarify the difference between CAM and immunosuppressive drugs such as FK506 in vitro (30). The article demonstrated that CAM could modify T-cell proliferation and IL-2 production. This showed that CAM has similar effects on FK506. However, they also showed that combined treatment with CAM (1.6 to 40 µg/ml) and FK506 (0.0001 to 0.001 µg/ml) resulted in an additional inhibition of T-cell proliferation. These results suggest that pharmacokinetics is significantly different, and there are different immunological receptors or signal cascades between CAM and FK506. Further studies would be needed to compare the immunological effects between CAM and conservative immunosuppressive agents.
Secondly, we demonstrated that CAM inhibited the development of graft arterial disease (GAD) formation in the MHC class II mismatch combination. Although GAD ultimately culminates in vascular stenosis and ischemic graft failure, the infallible care and prevention measures are still unknown. Therefore, it is noteworthy that CAM, which has been broadly used in clinical settings, can suppress the development of GAD that could not be prevented with conservative therapies. Chronic MHC class II mismatch allo-responses induce inflammatory and vascular wall cells to secrete growth factors that promote SMC intimal recruitment, proliferation, and matrix synthesis (31,32). Whereas pro-MMP-2 is basally produced by unstimulated SMCs in vitro, MMP-9 are produced after cytokine stimulation (33); MMP-9 is known to be required for SMC migration (34) and contributing to the intimal thickening hyperplasia of vascular lesion (14,35). The TIMP-1 is known as a natural inhibitor of MMP-9, and inhibition of MMP-9 by TIMP-1 is shown to decrease SMC migration and subsequent neointimal hyperplasia in the vascular injury model (36). The NF-B was well known as the regulator of the MMP-9 and -2; however, our data showed that CAM attenuated the expression of MMP-9 but not MMP-2 by Western blot and RT-PCR in macrophages and SMCs. The results might indicate indirect inhibition of NF-B activity by CAM treatment. In addition, our data showed that CAM attenuated the expression of MMP-9 without the alternation of TIMP-1; CAM might have an effect that directly inhibits the expression of MMP-9 mRNA without TIMP-1 activation. Previously, other investigators reported that erythromycin, one of the 14-member ring macrolides, suppressed the production of both MMP-9 and -2 in vitro (9,37). Interestingly, we demonstrated that CAM suppressed the production of MMP-9 but not MMP-2 in this report. Our data indicate that CAM has the potential to be a selective MMP-9 inhibitor. However, the detailed mechanism between CAM and MMP-9 has not yet been elucidated. Further investigation is needed. Although CAM inhibited GAD development and myocardial remodeling, graft function was not different between the groups in the MHC class II mismatch combination. Because murine graft function was evaluated by palpation in this study, this method might lack enough sensitivity to quantitatively evaluate graft function. An echocardiogram would be useful to analyze the function when it is applicable in future.
The dosage of CAM we used in this study (100 mg/kg/day) was approximately 10 times as large as the clinical dosage (10 to 20 mg/kg/day). Pharmacokinetics of CAM was different between mice and humans, as previously reported, and the various doses (5 to 600 mg/kg/day) of CAM could be used for murine examinations (16,17). Thus, we selected the dose for mice. However, further studies would be needed to determine an optimal dose-effect relationship.
We demonstrated for the first time that CAM suppressed myocardial inflammation and fibrosis through the attenuation of the inflammatory cell migration and myocardial cell infiltration by inhibition of MMP-9 activity, which resulted in suppression of acute cardiac rejection. In chronic rejection, CAM suppressed GAD development through the suppression of SMC proliferation and migration by MMP-9 inhibition. Although we focused on MMP-9 in this study, our results could not show direct evidence that CAM inhibition of MMP-9 was the primary effect of the drug. Therefore, further investigation would be needed to determine the importance of other factors that are modified by CAM.
In conclusion, CAM plays a significant role in the prevention of acute and chronic rejection through the inhibition of MMP-9 activity. Clarithromycin might be used in the suppression of transplant rejection and cardiovascular and other inflammatory diseases in clinical settings.
|
Acknowledgments
|
|---|
The authors thank Ms. Noriko Tamura and Ms. Yasuko Matsuda for their excellent technical assistance.
|
Footnotes
|
|---|
This work was supported by grants from the Japan Cardiovascular Research Foundation, a Grant-in-aid from the Japanese Ministry of Education, Science and Culture, a Grant-in-aid from the Japanese Ministry of Welfare, and the Organization for Pharmaceutical Safety and Research.
|
References
|
|---|
1. Isobe M, Yagita H, Okumura K, Ihara A. Specific acceptance of cardiac allograft after treatment with antibodies to ICAM-1 and LFA-1 Science 1992;255:1125-1127.[Abstract/Free Full Text]2. Suzuki J, Isobe M, Aikawa M, et al. Nonmuscle and smooth muscle myosin heavy chain expression in rejected cardiac allografts. A study in rat and monkey models. Circulation 1996;94:1118-1124.[Abstract/Free Full Text] 3. Taylor DO, Edwards LB, Boucek MM, et al. Registry of the International Society for Heart and Lung Transplantation: twenty-second official adult heart transplant report—2005 J Heart Lung Transplant 2005;24:945-955.[CrossRef][Medline] 4. Kikuchi T, Hagiwara K, Honda Y, et al. Clarithromycin suppresses lipopolysaccharide-induced interleukin-8 production by human monocytes through AP-1 and NF-kappa B transcription factors J Antimicrob Chemother 2002;49:745-755.[Abstract/Free Full Text] 5. Abe S, Nakamura H, Inoue S, et al. Interleukin-8 gene repression by clarithromycin is mediated by the activator protein-1 binding site in human bronchial epithelial cells Am J Respir Cell Mol Biol 2000;22:51-60.[Abstract/Free Full Text] 6. Leppert D, Waubant E, Galardy R, Bunnett NW, Hauser SL. T cell gelatinases mediate basement membrane transmigration in vitro J Immunol 1995;154:4379-4389.[Abstract] 7. Xia M, Leppert D, Hauser SL, et al. Stimulus specificity of matrix metalloproteinase dependence of human T cell migration through a model basement membrane J Immunol 1996;156:160-167.[Abstract] 8. Hibbs MS, Hoidal JR, Kang AH. Expression of a metalloproteinase that degrades native type V collagen and denatured collagens by cultured human alveolar macrophages J Clin Invest 1987;80:1644-1650.[Web of Science][Medline] 9. Hashimoto N, Kawabe T, Hara T, et al. Effect of erythromycin on matrix metalloproteinase-9 and cell migration J Lab Clin Med 2001;137:176-183.[CrossRef][Web of Science][Medline] 10. Ermolli M, Schumacher M, Lods N, Hammoud M, Marti HP. Differential expression of MMP-2/MMP-9 and potential benefit of an MMP inhibitor in experimental acute kidney allograft rejection Transpl Immunol 2003;11:137-145.[CrossRef][Web of Science][Medline] 11. Suzuki J, Isobe M, Kawauchi M, Endoh M, Amano J, Takamoto S. Altered expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in acutely rejected myocardium and coronary arteriosclerosis in cardiac allografts of nonhuman primates Transpl Int 2000;13:106-113.[CrossRef][Web of Science][Medline] 12. Yamani MH, Starling RC, Young JB, et al. Acute vascular rejection is associated with up-regulation of vitronectin receptor (alphavbeta3), increased expression of tissue factor, and activation of the extracellular matrix metalloproteinase induction system J Heart Lung Transplant 2002;21:983-989.[CrossRef][Web of Science][Medline] 13. Cho A, Reidy MA. Matrix metalloproteinase-9 is necessary for the regulation of smooth muscle cell replication and migration after arterial injury Circ Res 2002;91:845-851.[Abstract/Free Full Text] 14. Mason DP, Kenagy RD, Hasenstab D, et al. Matrix metalloproteinase-9 overexpression enhances vascular smooth muscle cell migration and alters remodeling in the injured rat carotid artery Circ Res 1999;85:1179-1185.[Abstract/Free Full Text] 15. Suzuki J, Ogawa M, Sagesaka YM, Isobe M. Tea catechins attenuate ventricular remodeling and graft arterial diseases in murine cardiac allografts Cardiovasc Res 2006;69:272-279.[Abstract/Free Full Text] 16. Tessier PR, Kim MK, Zhou W, et al. Pharmacodynamic assessment of clarithromycin in a murine model of pneumococcal pneumonia Antimicrob Agents Chemother 2002;46:1425-1434.[Abstract/Free Full Text] 17. Vallee E, Azoulay-Dupuis E, Swanson R, Bergogne-Berezin E, Pocidalo JJ. Individual and combined activities of clarithromycin and its 14-hydroxy metabolite in a murine model of Haemophilus influenzae infection J Antimicrob Chemother 1991;27(Suppl A):31-41. 18. Suzuki J, Koga N, Kosuge H, et al. Pitavastatin suppresses acute and chronic rejection in murine cardiac allografts Transplantation 2007;83:1093-1097.[CrossRef][Web of Science][Medline] 19. Kosuge H, Haraguchi G, Koga N, Maejima Y, Suzuki J, Isobe M. Pioglitazone prevents acute and chronic cardiac allograft rejection Circulation 2006;113:2613-2622.[Abstract/Free Full Text] 20. Wright JW, Brown TE, Harding JW. Inhibition of hippocampal matrix metalloproteinase-3 and -9 disrupts spatial memory Neural Plast 2007[E-pub ahead of print]. 21. Suzuki J, Ogawa M, Futamatsu H, Kosuge H, Tanaka H, Isobe M. A cyclooxygenase-2 inhibitor alters Th1/Th2 cytokine balance and suppresses autoimmune myocarditis in rats J Mol Cell Cardiol 2006;40:688-695.[CrossRef][Web of Science][Medline] 22. Kuzuya M, Kanda S, Sasaki T, et al. Deficiency of gelatinase a suppresses smooth muscle cell invasion and development of experimental intimal hyperplasia Circulation 2003;108:1375-1381.[Abstract/Free Full Text] 23. Kanai K, Asano K, Hisamitsu T, Suzaki H. Suppression of matrix metalloproteinase production from nasal fibroblasts by macrolide antibiotics in vitro Eur Respir J 2004;23:671-678.[Abstract/Free Full Text] 24. Ascher NL, Ferguson RM, Hoffman R, Simmons RL. Partial characterization of cytotoxic cells infiltrating sponge matrix allografts Transplantation 1979;27:254-259.[Web of Science][Medline] 25. Isobe M, Suzuki J. New approaches to the management of acute and chronic cardiac allograft rejection Jpn Circ J 1998;62:315-327.[CrossRef][Medline] 26. Mazzarella G, Petillo O, Margarucci S, Calabrese C, Peluso G. Role of monocyte/macrophage population in immune response Monaldi Arch Chest Dis 1998;53:92-96.[Medline] 27. Giannelli G, Falk-Marzillier J, Schiraldi O, Stetler-Stevenson WG, Quaranta V. Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5 Science 1997;277:225-228.[Abstract/Free Full Text] 28. Dubois B, Masure S, Hurtenbach U, et al. Resistance of young gelatinase B-deficient mice to experimental autoimmune encephalomyelitis and necrotizing tail lesions J Clin Invest 1999;104:1507-1515.[Web of Science][Medline] 29. Li Q, Verma IM. NF-kappaB regulation in the immune system Nat Rev Immunol 2002;2:725-734.[CrossRef][Web of Science][Medline] 30. Morikawa K, Oseko F, Morikawa S, Iwamoto K. Immunomodulatory effects of three macrolides, midecamycin acetate, josamycin, and clarithromycin, on human T-lymphocyte function in vitro Antimicrob Agents Chemother 1994;38:2643-2647.[Abstract/Free Full Text] 31. Hayry P. Chronic rejection: an update on the mechanism Transplant Proc 1998;30:3993-3995.[CrossRef][Web of Science][Medline] 32. Libby P, Tanaka H. The pathogenesis of coronary arteriosclerosis ("chronic rejection") in transplanted hearts Clin Transplant 1994;8:313-318.[Web of Science][Medline] 33. Galis ZS, Muszynski M, Sukhova GK, et al. Cytokine-stimulated human vascular smooth muscle cells synthesize a complement of enzymes required for extracellular matrix digestion Circ Res 1994;75:181-189.[Abstract/Free Full Text] 34. Kenagy RD, Hart CE, Stetler-Stevenson WG, Clowes AW. Primate smooth muscle cell migration from aortic explants is mediated by endogenous platelet-derived growth factor and basic fibroblast growth factor acting through matrix metalloproteinases 2 and 9 Circulation 1997;96:3555-3560.[Abstract/Free Full Text] 35. de Smet BJ, de Kleijn D, Hanemaaijer R, et al. Metalloproteinase inhibition reduces constrictive arterial remodeling after balloon angioplasty: a study in the atherosclerotic Yucatan micropig Circulation 2000;101:2962-2967.[Abstract/Free Full Text] 36. Lijnen HR, Soloway P, Collen D. Tissue inhibitor of matrix metalloproteinases-1 impairs arterial neointima formation after vascular injury in mice Circ Res 1999;85:1186-1191.[Abstract/Free Full Text] 37. Guo H, Lee JD, Yue H, et al. Effect of erythromycin on homocysteine-induced extracellular matrix metalloproteinase-2 production in cultured rat vascular smooth muscle cells Indian J Med Res 2005;121:764-770.[Web of Science][Medline]
|
Top
Abstract
Methods