Jun-ichi Suzuki

Gene Therapy for Attenuating Cardiac Allograft Arteriopathy Using Ex Vivo E2F Decoy Transfection by HVJ-AVE–Liposome Method in Mice and Nonhuman Primates

Cardiac allograft arteriopathy, which limits the long-term survival of recipients, is characterized by diffuse intimal thickening composed of proliferative smooth muscle cells. The transcription factor E2F plays a pivotal role in the coordinated transcription of cell-cycle regulatory genes. To test the hypothesis that double-stranded DNA with specific affinity for E2F (E2F decoy) is effective in preventing intimal hyperplasia, we performed ex vivo single intraluminal delivery of E2F decoy into cardiac allografts of mice and Japanese monkeys using the hemagglutinating virus of Japan (HVJ) artificial viral envelope–liposome method. In murine models, antisense cyclin-dependent kinase 2 (cdk2) kinase oligodeoxynucleotide (ODN) and no transfers were performed to compare the effects. Severe intimal thickening was observed, and multiple cell-cycle regulatory genes were enhanced in untreated allografts. E2F decoy prevented neointimal formation and suppressed these genes for up to 8 weeks, whereas antisense cdk2 kinase ODN had limited effects. In primate models, E2F decoy dramatically prevented neointimal thickening and suppressed multiple cell-cycle regulatory genes, whereas intimal thickening developed in the nontransfected or mismatch decoy-transfected allografts. Gel mobility shift assay proved the specific effects of E2F decoy, and reverse transcriptase–polymerase chain reaction documented that neither complication nor dissemination of HVJ into other organs was observed. We demonstrate that ex vivo gene delivery to allografts is a potent strategy to modify allograft gene expression, resulting in prevention of graft arteriopathy without systemic adverse effects.

An overview on Takayasu arteritis

SummaryTakayasu arteritis is a non-specific inflammatory disease of unknown etiology. It was first recognized as having a peculiar wreath-like arteriovenous anastomosis around the papillae of the retina by a Japanese ophthalmologist, Dr. M. Takayasu in 1908. A Japanese research committee reported more than 5,000 cases. For a supplement issue on Takayasu arteritis, this brief overview article has been written as an introduction to the disease.

Antisense Bcl-x oligonucleotide induces apoptosis and prevents arterial neointimal formation in murine cardiac allografts

OBJECTIVEnCardiac allograft arteriosclerosis, which limits long-term survival of recipients, cannot be prevented by conservative therapies. The arteriopathy is characterized by diffuse intimal thickening comprised of proliferative smooth muscle cells (SMCs). Cell death is a prominent feature of atherosclerosis; Bcl-x is one of the anti-apoptotic mediators.nnnMETHODSnTo test the hypothesis that antisense bcl-x oligodeoxynucleotide (ODN) is effective in preventing intimal hyperplasia through enhancing apoptosis after cardiac transplantation, we performed single intraluminal delivery of antisense bcl-x ODN into murine cardiac allografts (n = 9). DBA/2 (H-2d) hearts were transplanted into B10.D2 (H-2d) mice. Sense bcl-x ODN (n = 8) and no treatment (n = 8) studies were also performed.nnnRESULTSnAllografts were harvested at 4 weeks after transplantation; all allografts kept beating throughout the period. Coronary intimal thickening had developed in nontreated and sense ODN transfected allografts at 4 weeks after transplantation with enhanced expression of Bcl-x and cell adhesion molecules, and suppressed apoptosis. However, antisense bcl-x ODN prevented neointimal formation through enhanced apoptosis.nnnCONCLUSIONnThese results indicate that apoptosis of vascular SMCs induced by Bcl-x is associated with initial hyperplasia after heart transplantation. Antisense bcl-x ODN inhibits SMC proliferation by inducing apoptosis in graft coronary arteries.

Effects of intracellular cyclic AMP modulators on human eosinophil survival, degranulation and CD11b expression.

Background: Brochial asthma is characterized by infiltration of inflammatory cells such as lymphocytes and eosinophils. Theophylline is one of the most widely used drugs in the therapy of bronchial asthma, and phosphodiesterase (PDE) inhibition is thought to be an important mechanism of its anti–inflammatory actions. However, the detailed effects of PDE inhibition on eosinophils still remain unclear. Methods: Eosinophils in peripheral blood obtained from normal subjects and patients with mild off–season allergic rhinitis were purified using CD16 negative selection. The following effects of theophylline (nonselective PDE inhibitor), KF19514 (selective PDE IV inhibitor), mirlinone (selective PDE III inhibitor), procaterol (β2–adrenoceptor agonist) and N6, 2′–O–dibutyryladenosine 3′5′–cyclic monophosphate (dB–cAMP; AMP analogue) on eosinophils were examined: (1) survival in the presence of interleukin–5, (2) degranulation by granulocyte/macrophage colony–stimulating factor (GM–CSF) or platelet–activating factor (PAF), (3) CD11b expression under GM–CSF or PAF stimulation and (4) intracellular cAMP level. Results: Eosinophil survival was inhibited by theophilline, KF19514 or procaterol. GM–CSF– or PAF–induced degranulation was inhibited by theophylline, KF19514, procaterol or dB–cAMP. CD11b up–regulation by PAF was inhibited by theophylline, KF19514 or dB–cAMP, while GM–CSF–stimulated CD11b up–regulation was not significantly inhibited by any of the drugs tested. The levels of intracellular cAMP were increased by theophylline, KF19514 and procaterol. Conclusions: Intracellular cAMP is an important factor in the regulation of eosinophil biological functions. PDE IV inhibitors and β2–agonists are suggested to be useful for the treatment of bronchial asthma through inhibition of eosinophil effector function.

Improvement of eosinophilic heart disease after steroid therapy: successful demonstration by endomyocardial biopsied specimens.

SummaryA 62-year-old man with acute eosinophilic endomyocarditis developed congestive heart failure. The biopsy specimens revealed degranulated eosinophils and eosinophil cationic protein (ECP) in the endocardium, and in activated eosinophils and the myocardial interstitium. On electronmicroscopy, a characteristic cardiac myocytolytic change showing disruption at the intercellular junctional site was observed. After steroid treatment, clinical symptoms, systolic dysfunction and laboratory data dramatically improved. In the subsequent biopsy specimens, eosinophilic infiltration and ECP disappeared. Steroid therapy provided a beneficial effect in preventing the progression of cardiac damage. This effect was clearly documented by histochemical studies of the serial endomyocardial biopsy samples.

Expression of vascular endothelial growth factor and angiogenesis in cardiac myxoma: A study of fifteen patients

OBJECTIVEnTo clarify the association between angiogenesis and the clinicopathologic features in cardiac myxoma, vascular endothelial growth factor expression in the myxoma was examined by using reverse transcriptase polymerase chain reaction and immunohistochemistry, and the microvessel density was determined by counting microvessels in the myxoma by using immunostaining for platelet endothelial cell adhesion molecule 1.nnnMETHODSnSeven fresh-frozen and 15 formalin-embedded tissues were analyzed by means of reverse transcriptase polymerase chain reaction and immunostaining for vascular endothelial growth factor, respectively. The microvessel density was measured in the 15 formalin-embedded tissues. Furthermore, immunostaining for proliferating cell nuclear antigen was performed, and the proliferating cell nuclear antigen-labeling index was calculated.nnnRESULTSnAll of the 7 analyzed myxomas were positive for vascular endothelial growth factor messenger RNA, as determined by means of reverse transcriptase polymerase chain reaction, whereas atrial septum and atrium tissues were negative. Positive immunohistochemical reaction for vascular endothelial growth factor was observed in the cells of all 15 myxomas. The size of myxomas with high vascular endothelial growth factor expression was smaller than that of myxomas with low vascular endothelial growth factor expression. The microvessel density in myxomas with high vascular endothelial growth factor expression was higher than that in myxomas with low vascular endothelial growth factor expression. There was an inverse correlation between the tumor size and the ratio of the microvessel density in the central part to the microvessel density in the peripheral part of myxomas. Furthermore, there was an inverse correlation between the proliferating cell nuclear antigen-labeling index and the tumor size, and the prolferating cell nuclear antigen-labeling index in myxomas with high vascular endothelial growth factor expression was higher than that in myxomas with low vascular endothelial growth factor expression.nnnCONCLUSIONSnCardiac myxomas produce vascular endothelial growth factor, which probably induces angiogenesis for tumor growth.

The role of cell adhesion molecules in allograft rejection after penetrating keratoplasty in mice. Clinical and immunohistochemical study.

Abstract• Background: It has been reported that adhesion molecules play an important role in immunological rejection after organ transplantation. In the present study, we examined the role of ICAM-1/LFA-1 adhesion molecules in corneal allograft rejection and evaluated the immunological specificity of monoclonal antibodies (mAbs) in preventing allograft rejection in mice. • Methods: The allografted mice were intraperitoneally injected with 100 μg/day of the following mAbs: a control mAb, anti-ICAM-1 mAb, anti-LFA-1 mAb, or a mixture of anti-ICAM-1 and anti-LFA-1 mAbs from 1 day before to 7 days after surgery. The expression of ICAM-1 and LFA-1 molecules in the grafted cornea was studied immunohistochemically. The corneas from a syngeneic donor or a third-party strain were transplanted 4 weeks after the initial keratoplasty onto the mice treated with both anti-ICAM-1 and anti-LFA-1 mAbs. • Results: The allografts treated with anti-LFA-I mAb alone or both anti-ICAM-1 and anti-LFA-1 mAbs remained transparent for more than 2 weeks, and the survival rate at 8 weeks was 40% in both groups. ICAM-1 was expressed on the mononuclear cells, keratocytes and endothelial cells in the allografts without treatment. The second corneal grafts syngeneic to the initial donor remained transparent at 2 weeks, whereas those from the third party were rejected. • Conclusions: ICAM-1 and LFA-1 adhesion molecules play a crucial role in the pathophysiology of corneal transplant rejection. The immunosuppressive effects of anti-ICAM-1 and anti-LFA-1 mAbs are highly allospecific. The administration of mAbs to the adhesion molecules represents a new means of suppressing allograft rejection after penetrating keratoplasty.

Differential Th1 and Th2 cell regulation of murine cardiac allograft acceptance by blocking cell adhesion of ICAM-1/LFA-1 and VCAM-1/VLA-4

Administration of anti-intercellular adhesion molecule (ICAM)-1 monoclonal antibody (mAb) plus anti-lymphocyte function associated antigen (LFA)-1 mAb induces tolerance in murine cardiac transplantation, while anti-vascular cell adhesion molecule (VCAM)-1 mAb plus anti-very late antigen (VLA)-4 mAb administration prolongs graft survival, but leads to tolerance only in some cases. BALB/c mice hearts were transplanted into C3H/He recipients. Each combination of anti-VCAM-1 plus anti-VLA-4 mAbs (100 microg each/day, i.p.) or anti-ICAM-1 plus anti-LFA-1 mAbs (50 microg each/day, i.p.) was administered for 5 days. For control study, third group mice received daily with FK506 administration (1 mg/kg/day). The cardiac allografts and recipients spleens were harvested on day 7; the expression of cytokines were detected using immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and in situ RT-PCR. Th2 cytokines (IL-4 and IL-10) were markedly enhanced and Th1 cytokines (IFN-gamma and IL-2) were suppressed in recipients treated with anti-ICAM-1 mAb plus anti-LFA-1 mAb, while poor Th2 cytokine expression allowed persistent Th1 cytokine expression in recipient mice with anti-VCAM-1 mAb plus anti-VLA-4 mAb treatment. Both Th1 and Th2 cytokine expression was suppressed in FK506-treated mice. It is concluded that immunological tolerance and prolonged graft survival induced by blocking cell adhesion is regulated by different cytokine expression.

Intercellular adhesion molecule-1 on eosinophils is involved in eosinophil protein X release induced by cytokines.

Recent evidence suggests that adhesion molecules play important roles in eosinophil functions such as degranulation and superoxide anion production. CD11b/CD18 (Mac‐1) and CD49d/CD29 (VLA‐4) are involved in eosinophil–endothelial adhesion through their counterligands, intercellular adhesion molecule‐1 (ICAM‐1; CD54) and vascular cell adhesion molecule‐1 (VCAM‐1), respectively. CD54 is also induced on eosinophils by cytokine stimulation. We hypothesized that CD54 on human eosinophils may participate in eosinophil degranulation. CD54 was induced on eosinophils by a combination of human recombinant granulocyte–macrophage colony‐stimulating factor (rGM‐CSF) and human recombinant tumour necrosis factor‐α (rTNF‐α) within 2u2003hr of incubation, as determined by flow cytometric analysis. Recombinant GM‐CSF alone induced a slight but significant CD54 expression on eosinophils. Release of eosinophil protein X, an indicator of eosinophil degranulation, was induced by rGM‐CSF and this effect was synergistically enhanced by adding rTNF‐α. To determine the role of newly expressed CD54 in eosinophil degranulation, a blocking assay was performed using monoclonal antibody (mAb) against CD54 and CD18. Anti‐CD18u2003mAb and anti‐CD54u2003mAb markedly inhibited eosinophil degranulation induced by rGM‐CSF or a combination of rGM‐CSF and rTNF‐α. On the other hand, anti‐CD54u2003mAb had little effect on rGM‐CSF‐ or rGM‐CSF/rTNF‐α‐induced adhesion of eosinophils, whereas anti‐CD18u2003mAb significantly inhibited eosinophil adhesion. These results indicate that CD54 on eosinophils plays an important role in the eosinophil degranulation and that eosinophils are capable of interacting with other β2 integrin‐positive cells.

Enhanced Embryonic Nonmuscle Myosin Heavy Chain Isoform and Matrix Metalloproteinase Expression in Aortic Abdominal Aneurysm With Rapid Progression

Abdominal aortic aneurysms (AAAs) are characterized by structural deterioration of aortic wall leading to progressive dilatation. The histopathological changes in AAAs are particularly evident within the elastic media, which is normally comprised mainly of vascular smooth muscle cells (SMCs). There are vascular myosin heavy chain (MHC) isoforms; SM2 is specifically expressed in differentiated SMCs and SMemb is a nonmuscle-type MHC abundantly expressed in SMCs of the fetal aorta with an immature phenotype. Although AAA altered expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), pathophysiological role of SMC phenotypic modulation in the AAA progression remains uncertain. To determine whether phenotypic modulation in vascular SMCs contributes to arterial medial degeneration, we examined MHC expression in SMCs of AAA. Aortic specimens were obtained from patients with slowly progressed AAA (n = 12) and rapidly progressed AAA (n = 5), and compared with normal aortic tissue (n = 3). Immunohistochemical staining was performed for detection of SMemb, SM2, MMP (types 2 and 9) and TIMP (types 1 and 2). Faint SMemb and abundant SM2 were observed in normal aorta, while the balance shifted to SMemb predominance in AAAs. Compared with slowly progressed AAA tissue, rapidly expanded AAA tissue demonstrated marked increases in SMemb expression with suppressed SM2. Predominant SMemb expression indicates presence of phenotypic modulated SMCs and enhanced MMP; while abundant TIMP was seen in mature SMCs expressing SM2. SMemb expression is markedly increased in AAA with MMP enhancement, and a significant imbalance between SMemb and SM2 results in rapid progression of AAA.