Katsuaki Tsukioka

Suppression of acute and chronic rejection by hepatocyte growth factor in a murine model of cardiac transplantation: Induction of tolerance and prevention of cardiac allograft vasculopathy

Background—Although treatment with immunosuppressive agents has contributed to overcoming acute rejection and improving the midterm survival of transplanted hearts, cardiac allograft vasculopathy (CAV) has remained the main cause of primary graft failure. Recent approaches have shown that hepatocyte growth factor (HGF) exhibits cardiotrophic functions. We therefore addressed whether HGF would regulate acute and chronic rejection in cardiac transplantation. Methods and Results—We used a murine heterotopic cardiac transplantation model between fully incompatible strains and administered 500 &mgr;g · kg−1 · d−1 HGF during the initial 14 days after transplantation. The HGF-treated allografts showed significantly prolonged survival (42.3±4.1 days, P<0.001) compared with the controls (11.1±0.6 days), with tolerance induction in 47.4%. Histopathologically, the number of infiltrating cells was significantly decreased and myocardial necrosis was less prominent with a reduction of apoptosis in the allografts by HGF treatment during acute rejection. In the long-term surviving allografts, HGF significantly inhibited the development of CAV and interstitial fibrosis. With respect to intragraft cytokine mRNA expression, HGF treatment reduced the early expression of interferon-&ggr; and enhanced the expression of transforming growth factor-&bgr;1 during the acute phase and of interleukin-10 continuously through the acute phase to the chronic phase. Conclusions—Our findings demonstrate that HGF can prolong the survival of allografts by its cardioprotective and immunomodulative potencies. Thus, HGF administration may constitute a new therapeutic approach to preventing cardiac graft failure that has not been overcome by conventional immunosuppressive agents.

Expression of matrix metalloproteinases in cardiac allograft vasculopathy and its attenuation by anti MMP-2 ribozyme gene transfection

OBJECTIVE Proliferation and migration of vascular smooth muscle cells (SMCs) causes intimal thickening during cardiac allograft vasculopathy (CAV). This process requires the degradation or remodeling of extracellular matrix (ECM) surrounding the cells. Imbalance between degradation and accumulation of ECM also contributes to the development of CAV. In this study, we investigated the contribution of matrix metalloprotenases (MMPs), enzymes regulating ECM turnover, to the development of CAV. METHODS Donor hearts from male DBA mice were heterotopically transplanted to male B10.D2 recipient mice, and harvested at days 15 and 30 post transplantation. We examined expression MMP-2, -3, -9 and -13 of graft vessels using immunohistochemistry. To clarify the role of MMP-2 in CAV, anti MMP-2 ribozyme was delivered into donor hearts just before transplantation, mediated by a hemagglutinating virus of Japan-liposome complex to specifically suppress MMP-2 activity. RESULTS All MMPs were immunopositive in SMCs from the slightly thickened neointima at day 15. In the advanced stage of intimal thickening at day 30, in addition to increased number of SMCs, accumulation of collagenous fibers was observed; expression of MMP-3, -9 and -13 was decreased. In contrast, MMP-2 expression remained distinctly positive throughout the progression of the vascular remodeling. After the gene transfer of MMP-2 ribozyme, luminal occlusion was significantly decreased compared to non-treated allografts [25.0+/-6.5 vs. 55.1+/-7.0% (P<0.05)] at day 30 post transplantation. CONCLUSION MMP-2 is a principle MMP throughout the progression of the vascular remodeling in CAV. Anti MMP-2 therapy could therefore be one of the candidates for a supplemental therapy for CAV.

Expression of membrane-type 1 matrix metalloproteinase in coronary vessels of allotransplanted primate hearts.

BACKGROUND The mechanisms of intimal thickening in cardiac allograft vasculopathy (CAV) remain controversial after heart transplantation. Matrix metalloproteinase-2 (MMP-2) plays a crucial role in degrading extracellular matrix (ECM) during neointimal formation. Recently, it has been revealed that MMP-2 is activated by membrane-type 1 matrix metalloproteinase (MT1-MMP). This process involves tissue inhibitor of MMP-2 (TIMP-2), forming an MT1-MMP/TIMP-2/pro-MMP-2 complex. In this study, we hypothesize that these components contribute to the pathogenesis of CAV. METHODS Heterotopic cardiac allografting was performed in randomly paired Japanese monkeys with an immunosuppressive regimen of intravenous administration of antihuman CD18 monoclonal antibody. The donor hearts were harvested at Days 22, 28, 40, 41, and 95 posttransplantation. We examined expression of MMP-2, MT1-MMP, and TIMP-2 of graft vessels using immunohistochemistry and protein level by western blot analysis. RESULTS Pathologically, various degrees of neointimal formation were observed. In the allografts harvested at Days 22, 28, 40, and 41, MT1-MMP was expressed in the endothelial cells and smooth muscle cells (SMCs) in media of some arteries without histological change, accompanied by expression of MMP-2 and TIMP-2. In the severely thickened neointima of the allograft harvested at Day 95, MMP-2 and faint MT1-MMP were expressed in SMCs of severely thickened neointima and media; TIMP-2 expression was seen only in noncollagenous tissue of severely thickened neointima. MMP-2 protein was more intensely expressed in the allograft harvested at Day 95 than in the allograft harvest at Day 41, while TIMP-2 protein level was almost same in the 2 samples. CONCLUSION We observed the simultaneous expression of MMP-2, MT1-MMP, and TIMP-2. Thus, ECM degradation triggered by MT1-MMP/TIMP-2/pro-MMP-2 complex could be a novel mechanism of CAV.

Early growth-response factor 1 and basic transcriptional element-binding protein 2 expression in cardiac allografts.

Early growth-response factor 1 (Egr-1) and basic transcriptional element-binding protein 2 (BTEB2) are transcriptional factors that regulate multiple genes involved in phenotypic changes of smooth muscle cells (SMCs), one of the outstanding pathologic features of chronic cardiac allograft rejection. In this study, we used a heterotopic abdominal heart transplant model in monkeys to evaluate the roles of these molecules in graft coronary vasculopathy. We demonstrated that Egr-1 and BTEB2 are induced in vascular SMCs of rejected cardiac allografts well before morphologic changes, such as intimal thickening. These findings suggest that expression of Egr-1 and BTEB2 is one of the initial events in allograft angiopathy.

A case of inferior mesenteric artery aneurysm with an occlusive disease in superior mesenteric artery and the celiac artery.

A 72 year-old man was referred to our hospital for treatment of a gradually expanding inferior mesenteric artery (IMA) aneurysm associated with an occluded superior mesenteric artery (SMA) and a severely stenosed celiac artery (CA). Using 64-slice computer tomography (CT), we were able to accurately define a precise collateral visceral circulation from the IMA to the SMA and the CA, greatly clarifying preoperative strategy. The aneurysm was subsequently removed, with revascularization of the SMA and the CA accomplished through grafts from the abdominal aorta using 6 mm polytetrafluoroethylene (PTFE) grafts.

Egr-1 in vascular smooth muscle cell proliferation in response to allo-antigen.

BACKGROUND Early growth response factor-1 (Egr-1) plays an important role in regulating multiple factors involved in the progression of vascular lesions. This study examined our hypothesis that Egr-1 plays a critical role in the early stage of chronic cardiac allograft rejection and in the proliferation of the smooth muscle cell response to alloantigen. MATERIALS AND METHODS Antisense Egr-1 oligodeoxynucleotide (ODN) was ex vivo gene transfected into the donor hearts from DBA/2 mice, followed by heterotopic allografting into B10.D2 recipients. The allografts were harvested on day 30. Egr-1 and its target molecules, such as platelet-derived growth factor (PDGF)-A, basic fibroblastic growth factor (bFGF), vascular cell adhesion molecule (VCAM)-1, transforming growth factor (TGF)-beta and nonmuscle myosin heavy chain B (SMemb), were identified immunohistochemically, and the percentage of the lumen occluded by the intima was calculated. For the cell proliferation assay, sensitized T cells were harvested from B10.D2 recipients as stimulator and then added to the SMCs, which were harvested from DBA/2 mouse aorta. Cellular proliferation was measured and Egr-1 and its target gene expression were examined by real-time RT-PCR. RESULTS Egr-1 and its target genes were expressed in the thickened intima from untreated recipients. Egr-1 antisense ODN inhibited not only Egr-1 expression but also its target genes and significantly suppressed intimal thickening of coronary arteries. Egr-1 antisense ODN also significantly inhibited cell proliferation and expressions of Egr-1 and Egr-1 target genes in a mixed cell culture model. CONCLUSION We conclude that Egr-1 plays an important role in the formation of the cardiac allograft vasculopathy responding to alloantigens.

Mycotic Iliac Artery Aneurysm Caused by Clostridiumdifficile in a Patient with Axillobifemoral Bypass for Leriche Syndrome

A 74-year old man on hemodialysis developed a mycotic aneurysm caused by Clostridium difficile. To the best of our knowledge, this is only the second case of such an aneurysm reported in the literature. He had previously undergone axillobifemoral bypass grafting because of symptomatic infrarenal aortic stenosis. Although no blood flow was detected in his occluded right common iliac artery, it expanded rapidly despite intensive antibiotic therapy. As the blood supply to the lower limbs was already secured, only resection of the infected arteries was performed.

Expression of the transcriptional factor Egr-1/BTEB2 in cardiac xenograft vascular remodeling

IT IS important to consider that phenotypic change of smooth muscle cells (SMCs) is one of the important factors involved in vascular remodeling in cardiovascular diseases such as atherosclerosis and restenosis after balloon angioplasty. Phenotypic change of SMCs induces fibrosis of vessels by collagen synthesis, degradation of vessels by depletion of extracellular matrix, or intimal thickening by proliferation and migration of SMCs. In xenograft rejection, phenotypic change of SMCs plays an important role in the process of forming vascular lesion formation, as it does in common other cardiovascular diseases. However, little is known about this mechanisms. Early growth response factor-1 (Egr-1) and basic transcriptional element binding protein-2 (BTEB2) regulate multiple genes involved in the remodeling of vessels in various vascular diseases. Egr-1 is a transcriptional factor of BTEB2, which is itself a transcriptional factor regulating nonmuscle type myosin heavy chain (SMemb), which is expressed in embryonic SMCs of fetal aorta and proliferating SMCs. Therefore, Egr-1 and BTEB2 play an important role in the dedifferentiation of SMCs. It would be useful to consider expression of Egr-1 and BTEB2 in cardiac xenografts to investigate the mechanisms of xenotransplant rejection.

A Case of Traumatic Retrograde Type A Aortic Dissection Accompanied by Multiorgan Injuries

A 75-year-old woman was involved in a traffic accident and suffered retrograde type A aortic dissection, multiple rib fractures, and grade II hepatic injury accompanied by intraperitoneal bleeding. We performed total arch replacement using an open stent graft with cardiopulmonary bypass and circulatory arrest. This procedure requires anticoagulation and hypothermia, which are principally contraindicated in severe trauma patients. However, this situation was resolved by managing the patient non-operatively for 7 days, confirming the stabilization of other injured organs, and then performing the surgery. She required prolonged postoperative rehabilitation; however, she recovered steadily.

Nonmuscle myosin heavy chain and nuclear factor-kappa B expression in pig-to-primate xenotransplanted hearts.

IT IS well known that angiopathy on the intima of the blood vessels causes atherosclerosis. The damage on the intima injures endothelial cells and then smooth muscle cells (SMCs) of the media give rise to hypertrophy. The phenotypic change of SMCs should be necessary for the hypertrophy of the SMCs. Nonmuscle type myosin heavy chain (SMemb), which is an embryonic isoform of myosin heavy chain, is specifically expressed in fetal aorta and proliferating SMCs. Nuclear factor-kappa B (NF-k B) regulates multiple genes involved in vascular remodeling as a transcriptional factor. SMemb and NF-k B should be the important factors for the dedifferentiation and phenotypic change of SMCs. In the process of xenograft rejection, the same phenomenon as angiopathy has been reported and proliferating SMCs have been observed in blood vessels of rejected tissues. In this study, the expression pattern of SMemb and NF-k B in pig-toprimate xenotransplanted hearts were investigated. It will be useful to understand the mechanisms of xenograft rejection, particularly in cardiovascular remodeling.