Kentaro Setoyama

HMGB1 release in co-cultures of porcine endothelial and human T cells

Abstract:  High mobility group box‐1 (HMGB1) protein, primarily from the nucleus, is released into the extracellular milieu either passively by necrotic or damaged cells, or actively by secretion from monocytes/macrophages. Extracellular HMGB1 acts as a potent inflammatory stimulator by promoting cytokine (for example, tumor necrosis factor‐α) production, and also has pro‐coagulant activity. The signaling pathway initiated by receptor for advanced glycation end‐product (RAGE), which is the HMGB1 receptor, also induces complement activation. Recent studies have implicated HMGB1 in acute cardiac allograft rejection, and have identified infiltrating T cells and other damaged cells as its main sources. HMGB1 blockade using the anti‐HMGB1 antibody HMGB1 box‐A (amino‐terminal region) and soluble RAGE rescues mice from acute rejection. We therefore studied the release of HMGB1 in co‐cultures of porcine aortic endothelial cells (PAEC) and human leukocytes. Human T cells, but not B cells, monocytes or neutrophils, stimulated significant HMGB1 release in culture with PAEC; this activity required cell–cell contact and was dose‐dependent, as determined by Western blotting. The released HMGB1 originated from both cell types, as immunofluorescent microscopy showed that it was present in the cytosol of PAEC in contact with T cells, and had disappeared from the T‐cell nuclei. These results demonstrate that direct interactions between PAEC and T cells might be a key factor in triggering HMGB1 release, which suggests that HMGB1 is associated with graft rejection in the early phase.

Carbon monoxide reduces pulmonary ischemia–reperfusion injury in miniature swine

OBJECTIVES Carbon monoxide is produced endogenously as a by-product of heme catalysis and has been shown to reduce ischemia-reperfusion injury in a variety of organs in murine models. The aims of this translational research were to establish an in situ porcine lung model of warm ischemia-reperfusion injury and to evaluate the cytoprotective effects of low-dose inhaled carbon monoxide in this model. METHODS Warm ischemia was induced for 90 minutes by clamping the left pulmonary artery and veins in 8 Clawn miniature swine (Japan Farm CLAWN Institute, Kagoshima, Japan). The left main bronchus was also dissected and reanastomosed just before reperfusion. Four animals were treated with inhaled carbon monoxide at a concentration of approximately 250 ppm throughout the procedure. Lung function and structure were serially accessed via lung biopsy, chest x-ray films, and blood gas analysis. RESULTS Carbon monoxide inhalation dramatically decreased the lung injury associated with ischemia and reperfusion. Two hours after reperfusion, the arterial oxygen tension of the carbon monoxide-treated group was 454 +/- 34 mm Hg, almost double the arterial oxygen tension of the control group (227 +/- 57 mm Hg). There were fewer pathologic changes seen on chest x-ray films and in biopsy samples from animals in the carbon monoxide-treated group. Animals in the carbon monoxide-treated group also had fewer inflammatory cell infiltrates and a markedly smaller increase in serum concentrations of the proinflammatory cytokines interleukin 1beta, interleukin 6, and high-mobility group box 1 after ischemia-reperfusion injury. CONCLUSIONS The perioperative administration of low-dose inhaled carbon monoxide decreases warm ischemia-reperfusion injury in lungs in miniature swine. This protective effect is mediated in part by the downregulation of proinflammatory mediators.

Beneficial effects of perioperative low-dose inhaled carbon monoxide on pulmonary allograft survival in MHC-inbred CLAWN miniature swine.

Background. We have recently reported that perioperative low-dose carbon monoxide (CO) inhalation decreases lung ischemia-reperfusion injury in miniature swine. The aims of this study were to establish a large animal model of pulmonary allograft rejection using polymerase chain reaction-typed major histocompatibility complex (MHC)-inbred CLAWN miniature swine and to examine the effects of CO on allograft survival. Methods. Eleven CLAWN miniature swines received fully MHC-mismatched lungs followed by 12 days of tacrolimus (days 0–11; blood level 35–45 ng/mL). Six recipients received tacrolimus alone (control group). Five recipients were additionally treated with inhaled CO (180 min for donors until graft harvest; 390 min for recipients until 2 hr after reperfusion). Results. All recipients treated with tacrolimus alone uniformly rejected their grafts by postoperative day 63 with development of cytotoxic antidonor antibodies. CO treatment was effective in prolonging allograft survival from a mean of 47±7 to 82±13 days (P=0.017), with one CO-treated animal maintaining function until postoperative day 120. Development of antidonor antibodies and donor-specific responsiveness by cell-mediated lympholysis and mixed lymphocyte reaction assays was delayed in animals that received CO therapy. Furthermore, serum concentrations of proinflammatory cytokines (interleukin-1&bgr; and -6) 1 day after transplant were significantly decreased in the CO-treated group. Conclusions. Fully MHC-mismatched lungs in CLAWN miniature swine were consistently rejected within 63 days, suggesting that this is a robust large animal model ideal for investigating mechanisms and treatment of lung rejection. Perioperative low-dose CO inhalation prolonged graft survival and inhibited antidonor antibody production and was associated with decreased proinflammatory mediators in this model.

Hepatocyte growth factor sustains T regulatory cells and prolongs the survival of kidney allografts in major histocompatibility complex-inbred CLAWN-miniature swine.

Background. Although 12 days of high dose of FK506 permits the induction of tolerance of fully major histocompatibility complex (MHC)-mismatched allogeneic kidneys in MGH-miniature swine, we found that the same dose of FK506 is insufficient to induce such tolerance CLAWN-miniature swine. The CLAWN swine model was therefore chosen to study the potential immunoregulatory effects of human-recombinant hepatocyte growth factor (HGF). Methods. Ten CLAWN miniature swine received fully MHC-mismatched kidneys with 12 days (days 0–11) of FK506. Among these 10 recipients, 4 received 7 or 14 days of human-recombinant HGF starting at day 11. Graft function was assessed by daily serum creatinine and biopsies. Immunologic assays, including CD4/CD25 DP and FoxP3+ cells and development of antidonor antibodies, were performed. Results. Without HGF, all six CLAWN recipients developed severe acute rejection (Cre >9 mg/dL) within 3 weeks of transplantation. In contrast, in the four animals that received HGF for 7 to 14 days, stable renal function was observed for more than 50 days, although all grafts were ultimately rejected by postoperative day 80. Percent FoxP3+ cells in the CD4+CD25+ double positive population (T regulatory cells) in peripheral blood monocyte cells decreased in recipients with FK506 induction monotherapy while no reduction was observed in recipients treated with FK506 and HGF. Conclusion. This study demonstrates that in CLAWN swine treated with a dose of FK506 insufficient to induce tolerance across a fully MHC mismatched barrier, a short course of HGF may inhibit acute rejection while maintaining T regulatory cells. To our knowledge, this study provides the first evidence in a large animal transplantation model of HGFs immunoprotective effects.

Hepatocyte growth factor improves the survival of rats with pulmonary arterial hypertension via the amelioration of pulmonary hemodynamics

Hepatocyte growth factor (HGF) is a multifunctional growth factor with mitogenic, anti-apoptotic and anti-fibrotic activities. In this study, we investigated the effect of administration of recombinant human HGF on pulmonary arterial hypertension. Pulmonary arterial hypertension was induced in rats by a single injection of monocrotaline (MCT) and recombinant human HGF (0.12 mg/day) was administered into the right ventricle cavity using osmotic pumps, which were implanted subcutaneously 21 days after MCT injection. Continuous intravenous delivery of recombinant human HGF for 14 days led to prolonged survival of animals suffering from severe MCT-induced pulmonary arterial hypertension. Although a bolus injection of recombinant human HGF did not affect pulmonary arterial pressure, a 14-day administration of recombinant human HGF attenuated the inflammatory cell infiltrate, matrix accumulation and vascular medial thickening. As a consequence, the pulmonary lumen was enlarged and the pulmonary arterial pressure was significantly reduced. Additionally, continuous administration of recombinant human HGF suppressed lung tissue expression of platelet-derived growth factor, which plays an important role in the development of pulmonary arterial hypertension. These results indicate that recombinant human HGF possibly has a great potential for improving symptoms and altering the clinical course of pulmonary arterial hypertension.

Protective effect of neutralization of the extracellular high-mobility group box 1 on renal ischemia-reperfusion injury in miniature swine.

Background Strategies that reduce ischemia-reperfusion injury (IRI) have the potential to expand the numbers of available organs for transplantation. Recent reports in rodent models have demonstrated that high-mobility group box 1 (HMGB1) acts as an alarm in initiating the inflammatory response resulting from ischemic injury. The aim of this study was to evaluate the cytoprotective effects of anti-HMGB1 antibodies on renal IRI in preclinical large animals. Methods One hundred twenty minutes of warm and 60 min of cold renal ischemia were induced in 8 CLAWN miniature swine. Three of eight animals received intravenous anti-HMGB1 antibody at 1 mg/kg just before the reperfusion of renal blood flow. Renal function was assessed by serum creatinine and renal biopsy. Serum levels of interleukin (IL)-1&bgr;, IL-6, and HMGB1 were measured. Results The concentration of HMGB1 increased as early as 30 min after reperfusion and before the elevation of IL-1&bgr; and IL-6. Serum creatinine levels were markedly elevated, peaking at a median of 5 days (peak creatinine levels: 11.6±1.6 mg/dL) and recovering by day 14. Anti-HMGB1 antibody injection dramatically decreased renal damage as well as serum levels of HMGB1 associated with IRI. Renal function returned to near normal by day 9, and peak creatinine levels were markedly lower (7.4±0.2 mg/dL), and biopsies possessed fewer pathologic changes when compared to the control group. Conclusion In this study, we demonstrated the beneficial effects of perioperative administration of anti-HMGB1 antibody in reducing renal IRI in a clinically relevant, large animal model.

Edaravone, a Synthetic Free Radical Scavenger, Enhances Alteplase-Mediated Thrombolysis

The combination of alteplase, a recombinant tissue plasminogen activator, and edaravone, an antioxidant, reportedly enhances recanalization after acute ischemic stroke. We examined the influence of edaravone on the thrombolytic efficacy of alteplase by measuring thrombolysis using a newly developed microchip-based flow-chamber assay. Rat models of embolic cerebral ischemia were treated with either alteplase or alteplase-edaravone combination therapy. The combination therapy significantly reduced the infarct volume and improved neurological deficits. Human blood samples from healthy volunteers were exposed to edaravone, alteplase, or a combination of alteplase and edaravone or hydrogen peroxide. Whole blood was perfused over a collagen- and thromboplastin-coated microchip; capillary occlusion was monitored with a video microscope and flow-pressure sensor. The area under the curve (extent of thrombogenesis or thrombolysis) at 30 minutes was 69.9% lower in the edaravone-alteplase- than alteplase-treated group. The thrombolytic effect of alteplase was significantly attenuated in the presence of hydrogen peroxide, suggesting that oxidative stress might hinder thrombolysis. D-dimers were measured to evaluate these effects in human platelet-poor plasma samples. Although hydrogen peroxide significantly decreased the elevation of D-dimers by alteplase, edaravone significantly inhibited the decrease. Edaravone enhances alteplase-mediated thrombolysis, likely by preventing oxidative stress, which inhibits fibrinolysis by alteplase in thrombi.

Application of a Novel Anti-Adhesive Membrane, E8002, in a Rat Laminectomy Model

Neuropathic pain after spinal surgery, so-called failed back surgery syndrome, is a frequently observed common complication. One cause of the pain is scar tissue formation, observed as post-surgical epidural adhesions. These adhesions may compress surrounding spinal nerves, resulting in pain, even after successful spinal surgery. E8002 is an anti-adhesive membrane. In Japan, a clinical trial of E8002 is currently ongoing in patients undergoing abdominal surgery. However, animal experiments have not been performed for E8002 in spinal surgery. We assessed the anti-adhesive effect of E8002 in a rat laminectomy model. The dura matter was covered with an E8002 membrane or left uncovered as a control. Neurological evaluations and histopathological findings were compared at six weeks postoperatively. Histopathological analyses were performed by hematoxylin–eosin and aldehyde fuchsin-Masson Goldner staining. Three assessment areas were selected at the middle and margins of the laminectomy sites, and the numbers of fibroblasts and inflammatory cells were counted. Blinded histopathological evaluation revealed that adhesions and scar formation were reduced in the E8002 group compared with the control group. The E8002 group had significantly lower numbers of fibroblasts and inflammatory cells than the control group. The present results indicate that E8002 can prevent epidural scar adhesions after laminectomy.

Uric acid enhances alteplase-mediated thrombolysis as an antioxidant

Uric acid (UA) therapy may prevent early ischemic worsening after acute stroke in thrombolysis patients. The aim of this study was to examine the influence of UA on the thrombolytic efficacy of alteplase in human blood samples by measuring thrombolysis under flow conditions using a newly developed microchip-based flow-chamber assay. Human blood samples from healthy volunteers were exposed to UA, alteplase, or a combination of UA and alteplase. Whole blood and platelet-rich plasma were perfused over a collagen- and thromboplastin-coated microchip, and capillary occlusion was monitored with a video microscope and flow-pressure sensor. The area under the curve (extent of thrombogenesis or thrombolysis) at 30 minutes was 92% lower in the UA–alteplase-treated group compared with the alteplase-treated group. D-dimers were measured to evaluate these effects in human platelet-poor plasma samples. Although hydrogen peroxide significantly decreased the elevation of D-dimers by alteplase, UA significantly inhibited the effect of hydrogen peroxide. Meanwhile, rat models of thromboembolic cerebral ischemia were treated with either alteplase or UA–alteplase combination therapy. Compared with alteplase alone, the combination therapy reduced the infarct volume and inhibited haemorrhagic transformation. UA enhances alteplase-mediated thrombolysis, potentially by preventing oxidative stress, which inhibits fibrinolysis by alteplase in thrombi.

Osteochondral Regeneration of the Loading-bearing Site Using a Scaffold Free Three-dimensional Construct of Adipose Tissue-derived Mesenchymal Stem Cells in Pigs

Background: Many surgical strategies for reconstruction of both bone and cartilage have ever been investigated to restore joint structure and function in the late stages of Osteoarthritis (OA). This study was designed to investigate the regeneration of articular cartilage and subchondral bone in the loading-bearing site using a three-dimensional (3D) construct of autologous adipose tissue-derived mesenchymal stem cells (AT-MSCs). Methods: A 3D construct consisting of approximately 1,920 spheroids each containing 5.0 × 104 AT-MSCs was implanted into an osteochondral defect (with a diameter of 6.8 mm and a depth of 6 mm) in the right femoral medial condyle in five adult mini-pigs. The contralateral (left femoral) defect was the control. At three and six months post-operatively, the defects were evaluated using both CT and MR imaging. The radiolucent volume (RV, mm3) of the defects was calculated based on the multiplanar reconstruction of the CT images. MR images and gross and histologic pathology features were scored using a modified-MOCART system and the ICRS system, respectively, at six months post-operatively. Results: The percentages of RVs at three and six months compared with those immediately after the surgeries were significantly decreased in the implanted defects compared with the control defects. The total scores of modified- MOCART system were also significantly increased in the implanted sites comparing to the controls. Although there were no statistical differences in the average of gross scores, the average histological scores were significantly higher in the implanted sites than in the control sites. Conclusion: This is the first report suggesting that implantation of a scaffold-free three dimensional construct of only AT-MSCs into the osteochondral defect regenerates the original cartilage and subchondral bone structures over six months post-operatively in the loading-bearing site of large animal.