Masahito Ogawa

Tea catechins improve left ventricular dysfunction, suppress myocardial inflammation and fibrosis, and alter cytokine expression in rat autoimmune myocarditis

Myocarditis is a clinically serious disease. Tea catechins have been shown to reduce inflammation; however the effects of catechins on the development of myocarditis have not been well studied.

Ultrasound-Microbubble–Mediated Intercellular Adhesion Molecule-1 Small Interfering Ribonucleic Acid Transfection Attenuates Neointimal Formation After Arterial Injury in Mice

OBJECTIVES The purpose of this study was to investigate the efficiency of small interfering ribonucleic acid (siRNA) in murine arteries. We transfected it using a nonviral ultrasound-microbubble-mediated in vivo gene delivery system. BACKGROUND siRNA is an effective methodology to suppress gene function. The siRNA can be synthesized easily; however, a major obstacle in the use of siRNA as therapeutics is the difficulty involved in effective in vivo delivery. METHODS To investigate the efficiency of nonviral ultrasound-microbubble-mediated in vivo siRNA delivery, we used a fluorescein-labeled siRNA, green fluorescent protein (GFP) siRNA, and intercellular adhesion molecule (ICAM)-1 siRNA in murine arteries. Murine femoral arteries were injured using flexible wires to establish arterial injury. RESULTS The fluorescein-labeled siRNA and GFP siRNA showed that this nonviral approach could deliver siRNA into target arteries effectively without any tissue damage and systemic adverse effects. ICAM-1 siRNA transfection into murine injured arteries significantly suppressed the development of neointimal formation in comparison to those in the control group. Immunohistochemistry revealed that accumulation of T cells and adhesion molecule positive cells was observed in nontreated injured arteries, whereas siRNA suppressed accumulation. CONCLUSIONS The nonviral ultrasound-microbubble delivery of siRNA ensures effective transfection into target arteries. ICAM-1 siRNA has the potential to suppress arterial neointimal formation. Transfection of siRNA can be beneficial for the clinical treatment of cardiovascular and other inflammatory diseases.

Novel IkB kinase inhibitors for treatment of nuclear factor-kB-related diseases.

Introduction: NF-κB is a key regulator of inflammation and immunity in cancer development. The IκB kinase (IKK) is a multisubunit complex containing catalytic subunits termed IKK-α, -β and -γ. It is well known that many pro-inflammatory stimuli require the IKK-β subunit for NF-κB activation. Areas covered: NF-κB affects the progression of inflammation-related diseases, such as myocardial ischemia, bronchial asthma, arthritis, cancer and other diseases. We review the characteristics and effects of these inhibitors on inflammatory and other diseases. Expert opinion: Various synthesized IKK inhibitors have been developed and they will be used clinically in the near future.

Pharmacological activation of the prostaglandin E2 receptor EP4 improves cardiac function after myocardial ischaemia/reperfusion injury

AIMS Increased expression of several subtypes of prostaglandin E(2) receptors (EP1-4) has recently been described in clinical and experimental myocardial ischaemia/reperfusion (I/R) injury. However, their pathophysiological significance in I/R remains obscure. Thus, we determined whether the activation of the prostanoid receptor, EP4, suppresses myocardial I/R injury. METHODS AND RESULTS To analyse the role of EP4, we administered an EP4 selective agonist (EP4RAG, 1 or 3 mg/kg) or vehicle to rats with myocardial I/R injury. After 7 days of reperfusion, I/R rats exhibited left ventricular (LV) dilatation and contractile dysfunction with myocyte hypertrophy and interstitial fibrosis. EP4RAG significantly reduced infarction area/ischaemic myocardium (72.4 +/- 0.7 vs. 23.3 +/- 0.6%; P < 0.05) and improved LV contraction and dilatation compared with that of the vehicle. EP4RAG also attenuated the recruitment of inflammatory cells, especially macrophages, and interstitial fibrosis in hearts. Monocyte chemoattractant protein (MCP)-1 and other cytokines were increased in both non-ischaemic (area not at risk, ANAR) and ischaemic (area at risk, AAR) myocardium; however, western blot analysis and RNase protection assay showed that EP4RAG suppressed these changes. Gelatin zymography revealed EP4RAG significantly reduced matrix metalloproteinase-2 and -9 activities in both ANAR and AAR. Chemoattractant assay demonstrated that EP4RAG suppressed the migration of cytokine-stimulated macrophages and decreased the level of MCP-1 production in the supernatant (587.3 +/- 55.3 vs. 171.5 +/- 47.5 pg/mL; P < 0.05). CONCLUSION The data suggest that the EP4 agonist is effective for attenuation of I/R injury by suppressing MCP-1 and the infiltration of inflammatory cells, especially macrophages.

Ultrasound-Microbubble-Mediated NF-κB Decoy Transfection Attenuates Neointimal Formation after Arterial Injury in Mice

Objective: Decoy transfection is a significant methodology for suppressing gene activation. The decoy can be synthesized easily; however, the major obstacle is the difficulty involved in effective in vivo delivery. Methods and Results: We used a fluorescein-labeled decoy to investigate the ultrasound-microbubble-mediated in vivo delivery in normal and injured mouse arteries. We showed that this approach could deliver the decoy into target tissues. In addition, we performed in vivo NF-ĸB decoy transfection into murine injured arteries using the ultrasound-microbubble method. Murine femoral arteries were injured using flexible wires to establish arterial injury. Pathologically, neointima/media areas in the NF-ĸB decoy transfection using ultrasound-microbubble group showed less than those in the control groups. Immunohistochemistry revealed that enhanced expression of inflammatory factors was observed in nontreated injured arteries, while the NF-ĸB decoy suppressed the expression. Conclusion: We revealed that ultrasound-microbubble delivery of the decoy is effective for transfection into target organs. We also indicated that NF-ĸB decoy transfection using this method has potential for the suppression of neointimal formation. Ultrasound-mediated transfection of the decoy can be beneficial for the clinical treatment of restenosis after coronary intervention and other cardiovascular diseases.

The Mechanism of Anti-inflammatory Effects of Prostaglandin E2 Receptor 4 Activation in Murine Cardiac Transplantation

Background. Prostaglandin E2 (PGE2) is a pathogenesis of inflammatory diseases; PGE2 plays a key role in association of anti-inflammation and immune suppression. EP4, which is a PGE2 receptor, is known to suppress the production of inflammatory cytokines and chemokines in vitro. Although it has been reported that EP4 agonists prolonged cardiac allograft survival, little has been elucidated the immunologic mechanism. Methods. We injected a selective EP4 agonist (EP4RAG) into recipient mice with heterotopic cardiac transplantation. Results. EP4RAG significantly prolonged the graft survival compared with the vehicle-treated group. Although the vehicle-treated group showed severe myocardial cell infiltration, the EP4RAG-treated group attenuated the development on day 7. EP4RAG suppressed various proinflammatory factors such as cytokines, chemokines, adhesion molecules, and nuclear factor-&kgr;B (NF-&kgr;B) compared with the vehicle-treated group. We also demonstrated that EP4RAG suppressed the activation of macrophages, but it did not affect to T lymphocytes in vitro. EP4RAG inhibited the activation of NF-&kgr;B compared with the control group. Conclusion. Pharmacological selective EP4 activation suppressed the production of proinflammatory factors by inhibition of NF-&kgr;B activity in cardiac transplantation.

Clarithromycin Attenuates Acute and Chronic Rejection Via Matrix Metalloproteinase Suppression in Murine Cardiac Transplantation

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.

Porphyromonas gingivalis promotes murine abdominal aortic aneurysms via matrix metalloproteinase-2 induction.

BACKGROUND AND OBJECTIVE Abdominal aortic aneurysm (AAA) is a common and lethal disorder, and MMPs are highly expressed in AAA lesions. Large numbers of periodontopathic bacteria have been reported to be present in specimens obtained from the aortic walls of patients with an AAA. The purpose of this study was to analyze the influence of periodontopathic bacteria on AAA dilatation. MATERIAL AND METHODS AAAs were produced in mice by the periaortic application of 0.25 M CaCl(2), and NaCl was used as a control. The mice were inoculated once weekly with live Porphyromonas gingivalis, live Aggregatibacter actinomycetemcomitans or vehicle. RESULTS Four weeks after the periaortic application of either CaCl(2) or NaCl, a significant increase was observed in the aortic diameter of P. gingivalis-challenged mice compared with the vehicle control mice (p < 0.05), whereas there was no statistically significant increase in the aortic diameter of the A. actinomycetemcomitans-challenged mice. Immunohistochemical analysis found significantly higher numbers of CD8-positive and MOMA2-positive cells and significantly higher levels of MMP-2 in the aneurysmal samples of P. gingivalis-challenged mice compared with control mice. Live P. gingivalis promoted a significant proliferation of splenocytes in comparison with P. gingivalis-lipopolysaccharide and live A. actinomycetemcomitans (p < 0.05). CONCLUSION These findings demonstrate that challenge with P. gingivalis, but not with A. actinomycetemcomitans, can accelerate, or even initiate, the progression of experimental AAA through the increased expression of MMPs.

Dietary consumption of green tea catechins attenuate hyperlipidaemia-induced atherosclerosis and systemic organ damage in mice

Objective — The effects of consuming green tea catechins on the development of hyperlipidaemiainduced systemic organ damage have not been well studied; we investigated the effect using low density lipoprotein receptor knockout mice. Methods and Results — Mice were treated with high cholesterol food containing 0.2 or 4% catechins and they were supplemented for 35 weeks. High plasma cholesterol levels, liver and renal dysfunctions were observed in no catechin fed mice, while chow containing catechin suppressed these levels and damages. Severe atherosclerosis of the aorta, fatty liver and renal injury were also shown in the control mice; inflammatory factors were enhanced in these lesions of nontreated mice.The lesions were attenuated with suppression of the inflammatory factors in the chow-contained catechin treatment group. Conclusion — Dietary consumption of tea catechins attenuated the development of the systemic organ damage; thus, this has a clinical effect against systemic inflammatory diseases.

Attenuation of experimental autoimmune myocarditis by blocking T cell activation through 4-1BB pathway

4-1BB, a member of the tumor necrosis factor receptor (TNFR) family, binds the 4-1BB ligand (4-1BBL), works as a costimulatory molecule, and regulates T cell-mediated immune responses. Although inflammation is an essential pathological feature of myocarditis, the role of 4-1BB in experimental autoimmune myocarditis (EAM) remains unclear. Lewis rats were immunized on day 0 with purified porcine cardiac myosin to establish EAM. 4-1BB-immunoglobulin (4-1BBIg) was administered intraperitoneally (n=6) a total of 9 times (3 times per week). Rats were killed on day 21 to study effects of 4-1BB/4-1BBL pathway blockade. For controls, isotype-matched human IgG was administered in other EAM rats (n=6). Histologic and echocardiographic examination showed development of EAM attenuated by 4-1BBIg. Suppression of mRNA expression for IL-1alpha, IL-1beta, IL-4, IL-6, and TNF-alpha was noted in the heart tissue treated with 4-1BBIg. Treatment with 4-1BBIg reduced production of Th1-type cytokines, and inhibited T cell proliferation in vitro. In the 4-1BB signaling pathway in splenocytes, 4-1BBIg suppressed JNK, p38, and IkappaB activity but not that of ERK1/2. Blockade of T cell activation through the 4-1BB/4-1BBL pathway regulates development of EAM; therefore, 4-1BB may be an effective target for treating myocarditis.