Jun-ichi Suzuki, Department of Advanced Clinical Science and Therapeutics, University of Tokyo, 7–3-1 Hongo, Bunkyo, Tokyo 113–8655, Japan. Tel.: +81-3-5800-9116; Fax: +81-3-5800-9182; E-mail: firstname.lastname@example.org
Macrolide antibiotics are broadly used for the treatment of various microbial infections. However, they are also known to have multiple biologic effects, such as alteration of inflammatory factors and matrix metalloproteinases (MMPs). Because of controversial results in clinical trials, the effects of macrolides on cardiovascular diseases are still to be elucidated. It has been reported that MMP activity is upregulated in various cardiovascular diseases, such as myocarditis, cardiac transplant rejection and myocardial infarction. However, little is known about the effects of macrolides on cardiovascular diseases. We have reported that clarithromycin suppressed the development of myocarditis, cardiac rejection and myocardial ischemia using animal models. In this article, we reviewed the roles of MMPs in cardiovascular diseases and the effects of macrolides on the prevention of adverse tissue remodeling.
Several studies have demonstrated that there is a significant association between chronic inflammation and atherosclerotic cardiovascular disease . Recent studies have accumulated evidence that suggests bacterial infection, such as periodontitis  and pneumonia , contribute to cardiovascular disease. Thus, antibiotic therapies might be useful in the prevention of cardiovascular events . Sinisalo et al. reported the effect of antibiotic therapy on the secondary prevention of acute coronary syndrome in the Clarithromycin in Acute Coronary Syndrome Patients in Finland (CLARIFY) trial. They randomly assigned the patients with acute non-Q-wave infarction or unstable angina to receive double-blind treatment with either clarithromycin or placebo for three months. The results showed that clarithromycin reduced the risk of ischemic cardiovascular events in patients with acute non-Q-wave infarction or unstable angina . They also revealed that long-term clarithromycin therapy was beneficial in the prevention of recurrent cardiovascular events in nonperiodontitis patients  and in patients with C4 deficiency . On the other hand, Jespersen et al. demonstrated that short-term clarithromycin therapy significantly increased cardiovascular mortality in patients with stable coronary heart disease . They also demonstrated that concomitant statin treatment in the stable patients with coronary heart disease abrogated increased cardiovascular mortality . Berg et al. tried to reveal the pathological mechanism of the controversial results of secondary prevention trials. However, clarithromycin did not change the levels of inflammatory markers (C-reactive protein, interleukin (IL)-2 receptor, IL-6, IL-8, and tumor necrosis factor [TNF]-alpha) in the patients with atherosclerosis . Originally, the CLARIFY investigators tried to exploit the antimicrobial effects of clarithromycin, however, their results did not exclude nonantimicrobial effects . Therefore, the effects of clarithromycin on cardiovascular diseases are still to be elucidated.
Clarithromycin suppresses transcription factors, such as activator protein (AP)-1 and nuclear factor-kappa B (NF-κB). The down-regulation of the transcription factors inhibited pro-inflammatory cytokine production [12,13] and matrix metalloproteinase (MMP) activity. It has been reported that MMP activity is upregulated in various cardiovascular diseases, such as myocarditis [14–17], cardiac transplant rejection [18,19], myocardial infarction (MI) [20,21], and abdominal aortic aneurysm [22,23]. However, little is known about the effect of clarithromycin on cardiovascular diseases via MMPs. In this article, we reviewed the roles of MMP activity in cardiovascular diseases and the potent effect clarithromycin has on the prevention of tissue remodeling.
MMPs and Cardiovascular Diseases
MMPs are a large family of proteinases that proteolytically degrade extracellular matrix (ECM). The degradation of ECM is an important event in the process of inflammation and tissue remodeling. MMPs are also involved in the regulation of cell behavior through the release of growth factors and cytokines from the substrates. This increases the magnitude of their effects . Inflammatory cells, such as lymphocytes and macrophages, produce MMPs [25,26], and their levels have been shown to be upregulated in inflamed hearts . MMPs also play an important role in cell migration, which results in tissue inflammation, and are also involved in the migratory capabilities of inflammatory cells .
A member of MMPs, MMP-9 (gelatinase B) has a pivotal role in tissue remodeling and the migration of smooth muscle cells (SMCs), macrophages and other cells . Thus, the gelatinase is critical in the process of infiltration in inflamed tissues. Previous papers showed that MMP-9 plays a crucial role in the development of acute myocarditis . In cardiac transplantation, the levels of MMP-9 have shown to be upregulated in grafts with human and various mammal organ transplantation [18,31]. T lymphocytes and macrophages are a major component of the cellular infiltration and are a large source of MMPs in allograft rejection [32,33]. They also contribute to the development of tissue injury in acute inflammatory reaction [28,33]. MI is also known to be a MMP-related cardiovascular disease. Because T lymphocytes and macrophages are a major component of the cellular infiltration after myocardial ischemia reperfusion injury, MMPs contribute to the development of tissue injury in acute inflammatory reaction [34–36]. Hakuno et al. found that MMP-2, MMP-13 and periostin expression levels were markedly increased in hearts of wild-type mice with a high-fat diet. Periostin increased MMP secretion from cultured valvular interstitial cells, endothelial cells and macrophages in a cell type-specific manner . Hansson et al. revealed that the serum tissue inhibitor of matrix metalloproteinase (TIMP)-1 was positively related to left ventricular mass and wall thickness. It was also inversely related to left ventricular ejection fraction. This may imply that ECM remodeling is already involved in the earliest stages of the process . During ECM remodeling in the myocardium, cardiac fibroblasts also play a pivotal role to mediate MMP/TIMP regulation. It is known that various T lymphocyte phenotypes differentially affect organ fibrosis and cardiac fibroblasts through modulating collagen and MMP/TIMP gene expression. Thus, the relative expression of TIMPs and MMPs altered cardiac collagen quantity, leading to alterations in ECM composition .
Dormán et al. showed the therapeutic potential of MMP inhibitors on the development of both chronic and acute diseases. They reviewed the novel MMP mechanisms, such as recent discoveries of oxidative/nitrosative activation and phosphorylation of MMPs. They also revealed nonmatrix related intra- and extracellular targets of MMPs. Traditionally, MMPs were only known as enzymes involved in chronic processes on a days-week time-scale. However, they also act acutely in response to oxidative stress on a minutes time-scale. Thus, MMPs play an important role not only in chronic (e.g., arthritis, chronic obstructive pulmonary disease and cancer metastasis) but also in acute (e.g., myocardial ischemia, vascular injury and septic shock) tissue remodeling [40,41]. Chow et al. reviewed the acute actions of both extracellular and intracellular targets of MMPs in ischemic hearts. Extracellular targets may include matrix proteins such as laminin, elastin, type IV collagen and fibronectin. On the other hand, oxidative stress-induced MMP-2 can rapidly cleave intracellular sarcomeric proteins such as TnI and MLC-1, causing acute contractile dysfunction. They concluded that a comprehensive understanding of MMP biology is necessary for the development of novel pharmacological therapies to suppress cardiovascular disease . Thus, MMPs play a critical role in cardiac remodeling that is caused by different etiologies.
To clarify the pathophysiology of MMPs in cardiovascular disease, genetically deficient mice have been used. Moshal et al. clarified that MMP-9 deficiency attenuated myocardial contractile dysfunction in heart failure induced by volume overload . On the other hand, there are multiple reports in various experimental animal models in which the absence of MMP-2 or MMP-9 expression led to worsened inflammation/infiltration and disease exacerbation. Campbell et al. reported on the importance of MMP-2 and MMP-9 in the pathogenesis of cardiac allograft rejection using MMP-2 or -9 deficient mice. They also showed that MMP-2-deficiency prolonged allograft survival time, while MMP-9-deficiency decreased allograft survival time . Cheung et al. demonstrated that coxsackievirus B3 resulted in a larger area affected by myocarditis in MMP-9-deficient mice than in wild-type mice . Despite the high therapeutic potential of MMP inhibitors, many clinical trials have failed to date. A majority of clinical trials using MMP inhibitors have been discontinued due to safety reasons (e.g., musculoskeletal side effects, etc.) . Such adverse events were not reported in patients treated with clarithromycin or other 14-member ring macrolides for noncardiac diseases. Because clarithromycin and other 14-member ring macrolides are not specific MMP inhibitors, these compounds act differently not to meet the same fate as failed specific MMP inhibitors. At this point, doxycycline is the only clinically applicable MMP inhibitor to treat periodontal disease .
Clarithromycin and MMPs
The 14-member ring macrolides are recognized as potent antibiotics for the treatment of various microbial infections [48–50]. Long-term 14-member ring macrolide therapy has been established for the clinical treatment of diffuse panbronchiolitis . One 14-member ring macrolide, clarithromycin, is known to be not only a potent antibiotic for the treatment of various microbial infections but also has multiple biologic effects, such as altering inflammatory factors [11–13] (Figure 1). It has been reported that clarithromycin also affects various cell types, such as macrophages  and neutrophils  (Table 1). While the 16-member ring macrolides do not have multiple effects, it is believed that the multiple effects are characteristic of 14-member ring macrolides . Kanai et al. tried to explain why 14-member ring macrolides had multiple effects but 16-member ring macrolides did not. They demonstrated that a 14-member ring macrolide, roxithromycin, could inhibit MMP–9 in vitro, while a 16-member macrolide, josamycin, did not exert suppressive effects on MMP-9 production. They speculated that 14-member ring macrolides could not suppress TIMP–2 production. Thus, the 14-member ring macrolide treatment inactivated MMP-9 . Doxycycline, which is a member of the tetracycline antibiotics group, is also known to directly inhibit MMP-9. Abdul-Hussien et al. reported that doxycycline reduced MMP-3 and MMP-25 mRNA expression, neutrophil collagenase and gelatinase protein levels in the aortic wall. They also concluded that doxycycline improved the proteolytic balance in AAA through aortic wall neutrophil content . These drugs are effective via antiinflammatory mechanisms rather than antibacterial activity.
It has been reported that clarithromycin inhibits the activation of NF-κB. Kikuchi et al. investigated the effects of clarithromycin on cytokine production using human peripheral monocytes and the monocytic leukaemia cell line, THP-1. Clarithromycin suppressed IL-8 production in a dose-dependent manner in both monocytes and THP-1 cells. An electromobility shift assay revealed that lipopolysaccharide (LPS) increased the specific binding of NF-κB, whereas clarithromycin suppressed it [12,13]. Because NF-κB is well known as a regulator of MMP-9, clarithromycin can suppress MMP-9 activity through NF-κB inactivation. This could result in attenuated myocardial inflammatory cell infiltration. This alteration consequently decreased myocardial remodeling compared to the control group. The presumed effects of clarithromycin on MMP regulation are known to be shared by other 14-member ring macrolides, such as erythromycin and roxithromycin. Guo et al. elucidated that erythromycin decreased homocysteine-induced MMP-2 secretion in vascular SMCs . Hashimoto et al. showed that expressions of MMP-9 protein and mRNA were downregulated by erythromycin in inflammatory cells . Similarly, Tabuchi et al. revealed that roxithromycin inhibits MMP-13 via the downregulation of Runx2 in human gingival epithelial cell cultures . Kanai also showed that roxithromycin significantly suppressed MMP-9 production from neutrophils in response to LPS stimulation . Hansson et al. revealed that serum TIMP-1 levels were related to LV mass, wall thickness, and inversely to systolic function in large population-based samples. Conversely, there was a weak relationship between MMP-9 and LV wall thickness . Thus, 14-member ring macrolide antibiotics do not only affect MMP-9, but may also regulate factors such as TIMPs. Although there was no established evidence, clarithromycin may have an effect on TIMP-1. Therefore, clarithromycin may affect MMP via TIMP regulation. Because the effects of 14-member ring macrolides on TIMPs are still to be clarified, the detailed mechanism between clarithromycin and MMP/TIMP has to be elucidated.
Clarithromycin and Specific Cardiovascular Diseases
The CLARIFY trial demonstrated that clarithromycin reduced the risk of ischemic cardiovascular events [5–7]. However, other clinical trials using clarithromycin therapy showed controversial results [8–10]. Thus, we chose clarithromycin to elucidate the association with cardiovascular disease.
Myocarditis is an inflammatory heart disease that is considered to be one of the causes of dilated cardiomyopathy . Although viral infection is the most common cause of human myocarditis, autoimmune myocarditis also occurs as a giant cell myocarditis [15,16]. A novel model of rat experimental autoimmune myocarditis (EAM) has been used to investigate the pathogenesis of myocarditis induced by autoimmune mechanisms [59–62]. T lymphocytes and macrophages are a major component of the cellular infiltration of EAM; the cells contribute to the development of tissue injury in acute inflammatory reaction . A recent study reported that lipocalin-2/neutrophil gelatinase-B associated lipocalin (Lcn2/NGAL) was expressed in hearts with myocarditis. Lcn2/NGAL expressing cells in EAM hearts were identified as cardiomyocytes, vascular wall cells, fibroblasts and neutrophils . Previous papers demonstrated that the conventional immunosuppressive drugs attenuated EAM. Zhang et al. showed that EAM was preventable by cyclosporine . They also highlighted that FK-506 prevented the progression of EAM  and the immunosuppressant was effective after onset of the disease . Because T lymphocytes and macrophages play a significant role in the initial progression of EAM, these immunosuppressive drugs attenuated EAM . After the inhibition of inflammatory cell activation, expression of immunological molecules by cardiomyocytes and inflammatory and interstitial cells were also suppressed . As Matsumoto reported, MMP-9 plays a crucial role in the development of myocarditis . Recently, we demonstrated that clarithromycin treatment negated the myocarditis-induced decrease in mean blood pressure compared to the untreated group. Echocardiogram revealed that clarithromycin prevented impairment of left ventricular contraction with less pericardial effusion. While nontreated EAM hearts demonstrated an increased heart per body weight ratio compared to that of native rats, clarithromycin administration significantly reduced the ratio compared to that of the nontreated EAM hearts. Pathologically, severe myocardial cell infiltration and fibrosis was observed in the nontreated hearts, while the affected lesions significantly decreased in the hearts treated with clarithromycin. Zymogram revealed that the MMP activity was markedly enhanced in the infiltrated area in the nontreated group, however, the clarithromycin-treated hearts attenuated the activity .
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 [18,19]. The allografts showed diffuse arterial neointimal formation (graft arterial disease, GAD) that consisted of SMCs, ECM, and various mononuclear leukocytes during long-term observation [18,72–79]. Although GAD ultimately culminates in vascular stenosis and ischemic graft failure, the measures to prevent GAD progression are still unknown .
We administered oral clarithromycin into murine cardiac allograft recipients to clarify its effect on heart transplantation. Total allomismatch and class II mismatch combinations were used to analyze graft survival and pathology. We revealed that clarithromycin significantly prolonged allograft survival, while nontreated allografts were acutely rejected in the major mismatch group. Pathologically, severe myocardial cell infiltration and fibrosis was observed in the nontreated group, while clarithromycin treatment significantly suppressed infiltration and fibrosis. The heavy neointimal thickening was observed in the coronary arteries of untreated allografts in this combination. However, clarithromycin attenuated intimal thickening. Immunohistochemically, clarithromycin markedly attenuated expression of CD4, CD8, CD11b, NF-κB p65, and MMP-9 while the expression was enhanced in the nontreated allografts. The mRNA levels of IFN-γ, IL-6, IL-10, IL-15 were significantly suppressed in the clarithromycin-treated group compared with those of the nontreated group. A gelatinase assay showed that MMP activity was markedly enhanced in the infiltrated area in the nontreated group; however, clarithromycin-treated grafts attenuated the activity .
Myocardial ischemia is a common antecedent event that predisposes a patient to congestive heart failure . Loss of cardiac function following myocardial ischemia occurs in the context of myocyte death and interstitial fibrosis, and that is referred to as ventricular remodeling . Recent studies have demonstrated that inflammatory responses may cause myocardial damage and fibrosis, leading to progressive impairment of cardiac function . Specifically, MMPs are important mediators in the pathogenesis of myocardial remodeling after ischemia . It is well known that the release and activation of MMPs significantly contribute to myocardial injury after ischemia and reperfusion. Lalu et al. showed that classical preconditioning inhibited ischemia/reperfusion-induced release and activation of MMP-2 . ECM of the heart is known to play an important role in LV remodeling. MMPs are responsible for ECM turnover and they are altered in cardiovascular pathologies, including MI and ischemic heart failure. It was reported that MMP inhibition prevented LV dilation and preserved cardiac function in animal models with an infarction. In spite of this, initial clinical trials with MMP inhibition post MI have failed. Dormán et al. speculated the reasons as (i) poor selectivity of the MMPIs, (ii) poor target validation for the targeted therapy, and (iii) poorly defined predictive preclinical animal models for safety and efficacy . Gallagher et al. reviewed the structural and functional roles of myocardial ECM, the evidence for MMP involvement in LV remodeling, and recent investigations into MMPs as prognostic markers and therapeutic targets . Giricz et al. showed that hyperlipidemia abolished preconditioning-induced inhibition of myocardial MMP-2 activation and release, and pharmacological inhibition of MMPs produces cardioprotection in both normal and hyperlipidemic rats .
Recently, we revealed that clarithromycin significantly reduced the infarction area and preserved ventricular contraction after myocardial ischemia in an animal experimental model. Pathologically, severe myocardial remodeling with enhanced MMP expression was observed in the nontreated group, while clarithromycin significantly suppressed these changes in hearts with myocardial ischemia. Zymography showed that clarithromycin reduced MMP-9 activity, while it was enhanced in the nontreated hearts. Immunohistochemical analysis showed that clarithromycin significantly reduced macrophage infiltration and MMP activity, while ischemia reperfusion resulted in ventricular remodeling with increased MMP activity and macrophage infiltration. Our data demonstrated that clarithromycin was effective in attenuating myocardial remodeling after ischemia by suppressing MMPs .
In this article, we reviewed MMP activity in cardiovascular diseases and the effect of clarithromycin treatment on the prevention of MMP-related tissue remodeling. We demonstrated that clarithromycin suppressed cardiac remodeling that is caused by myocarditis , transplant rejection , and myocardial ischemia . Furthermore, we highlighted that postinfarction remodeling, transplant rejection and myocarditis represent very different pathologic contexts in the initial steps. The initial steps were ischemia reperfusion injury in infarction, antigen presentation in transplantation, and autoimmunity in myocarditis. However, our previous papers demonstrated, at least in part, that these pathologic conditions included some common pathways during the development of ECM remodeling through MMP activation [71,81,87]. On the issue of myocarditis, Blauwet et al. made an editorial comment entitled “Antimicrobial agents for myocarditis: target the pathway, not the pathogen”. In his review, he commented that clarithromycin targeted the pathway of myocardial remodeling through MMP regulation, however, it did not target the EAM pathogen as an autoimmune reaction. Despite his comment being about myocarditis, “target the pathway, not the pathogen” is a common concept that can be applied to other diseases. Although an antigen presentation or an ischemic injury (pathogens) cannot be eliminated, clarithromycin suppresses MMPs (pathway), which is a cause of adverse remodeling (Figure 2). Because clarithromycin is not a specific MMP inhibitor, its effects on the injured heart may be due to actions not completely related to MMP inhibition. However, our previous papers demonstrated that its effects, at least in part, are related to MMP inhibition.
We have to discuss the issue regarding the long-term treatment with clarithromycin from a viewpoint of resistant bacteria against macrolides. Although long-term 14-member ring macrolide therapy has been established for the clinical treatment of diffuse panbronchiolitis , resistant bacteria against macrolides is still a serious issue. It was reported that H. pylori antibiotic resistance rates were 17.2% for clarithromycin; the prevalence rate of clarithromycin resistance increased from Europe to Asia, America and Africa . Kasahara et al. revealed that macrolide resistance profiles of microorganisms may be influenced according to the kind of macrolide antibiotics used . Therefore, we have to use different 14 member ring macrolides not to develop bacterial resistance. Because the effects of clarithromycin appear to be beneficial in the prevention of adverse tissue remodeling via MMP regulation, the 14 member ring macrolides are alternative treatments for suppressing chronic inflammatory remodeling. However, its detailed mechanism and long-term safety, including bacteria resistance, has not yet been elucidated in clinical settings. To be a future option for the treatment of cardiovascular diseases, derivatives of clarithromycin or other 14 member ring macrolides, which alter tissue MMP activity without antibacterial effects, will be needed.
In conclusion, MMP is critical for tissue remodeling in various cardiovascular diseases. Because of the anti-MMP activity of clarithromycin, the compound might be useful to prevent harmful cardiac remodeling in MMP related cardiovascular diseases.
This research is supported by the Japan Society for the Promotion of Science (JSPS) through its “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program).” We thank Ms. Noriko Tamura and Ms. Yasuko Matsuda for their excellent assistance.