Mst1 attenuates myocardial ischemia/reperfusion injury following heterotopic heart transplantation in mice through regulating Keap1/Nrf2 axis.

Ischemia reperfusion (I/R) injury remains a frequent adverse event that accompanies heart transplantation. Oxidative stress and aberrant production of free radicals were regarded as the culprit of cell death and tissue damage in post-transplant IR injury. Mst1 has been identified as a mediator of oxidative stress and Nrf2 regulates anti-oxidative enzymes, however, the interaction between Mst1 and Nrf2 anti-oxidative stress pathway remains to be clarified in the event of cardiac IR injury. Herein, the model of ischemia-reperfusion injury in heterotopic heart transplantation mice was firstly established.. We observed that cardiac IR induced upregulation of Mst1 and activation of Nrf2/HO-1pathway in mice receiving heterotopic heart transplantation. Further Cobalt dichloride-induced oxidative stress model of RAW264.7 macrophage cells were then established to mimic cardiac I/R injury, results showed that exposure to CoCl2 induced the upregulation of Mst1 and activation of Keap1/Nrf2 pathway, and genetic ablation of Mst-1 and inhibition of Keap1/Nrf2 pathway aggravated oxidative damage in those cells. Additional in vivo study showed that transfection of Mst1 shRNA spurred ROS generation and worsened cardiac damage in IR mice. Meanwhile, Mst1-KD mice receiving heart transplantation showed markedly downregulation of Nrf2, HO-1 yet upregulation of Keap1, indicating diminished protective effect against tissue damage caused by IR probably owing to the frustration of Keap1/Nrf2 pathway. Taken together, our findings demonstrated the protective effect of Mst1 from cardiac IR injury via triggering Keap1/Nrf2 axis and suppressing ROS generation, which shed light on the promising role of Mst1 in transitional management of IR injury resulted from cardiac transplantation.

Leflunomide Inhibits rat-to-Mouse Cardiac Xenograft Rejection by Suppressing Adaptive Immune Cell Response and NF-κB Signaling Activation.

Xenotransplantation is a potential solution for the severe shortage of human donor organs and tissues. The generation of humanized animal models attenuates strong innate immune responses, such as complement-mediated hyperacute rejection. However, acute vascular rejection and cell mediated rejection remain primary barriers to xenotransplantation, which limits its clinical application. In this study, we systematically investigated the immunosuppressive effect of LEF using a rat-to-mouse heart xenotransplantation model. SD rat xenogeneic hearts were transplanted into C57BL/6 mice, and survived 34.5 days after LEF treatment. In contrast, BALB/c allogeneic hearts were transplanted into C57BL/6 mice, and survived 31 days after LEF treatment. Compared to normal saline treatment, LEF treatment decreased xenoreactive T cells and CD19+ B cells in recipient splenocytes. Most importantly, LEF treatment protected myocardial cells by decreasing xenoreactive T and B cell infiltration, inflammatory gene expression, and IgM deposition in grafts. In vivo assays revealed that LEF treatment eliminated xenoreactive and alloreactive T and B lymphocytes by suppressing the activation of the NF-κB signaling pathway. Taken together, these observations complement the evidence supporting the potential use of LEF in xenotransplantation.

Vitamin D3 combined with antibody agents suppresses alloreactive memory T-cell responses to induce heart allograft long-term survival.

BACKGROUND: The pre-stored memory T cells in organ transplant patient carry a high risk of allograft rejection. The current study aimed to determine whether the allogenic response of adoptively transferred memory T cells in mice was suppressed by vitamin D3 monotherapy alone or in combination with monoclonal antibody treatment. METHODS: Prior to vascularized heterotopic heart transplantation, naïve C57BL/6 mice were primed with memory T cells. Recipient mice were administered vitamin D3 alone or in combination with monoclonal antibodies (anti-CD40L/ anti-LFA-1). Memory T cells and CD4+ forkhead box P3+ T cells in recipient spleens were measured using flow cytometry. Additionally, the expression of cytokines was measured by ELISA and quantitative PCR. Inflammatory factors in the grafts were identified by hematoxylin and eosin staining. RESULTS: Vitamin D3 in conjunction with anti-CD40L/ anti-LFA-1 antibodies were administered according to the median survival time from 6.5 to 80 days. The results revealed that grafts were protected through the prevention of inflammatory cell infiltration. Combined treatment decreased the mRNA levels of IL-2, IFN-γ and IL-10 and increased the mRNA levels of IL-4, Foxp3 and TGF-ß in the allograft. Rejection was suppressed by a reduction of CD4+CD44high CD62L+ and CD8+ CD44high CD62L+ memory T cells, the induction of regulatory T cells in the recipient spleen and a reduction of serum IL-2, IFN-γ and IL-10 levels. CONCLUSION: Vitamin D3 efficiently protected allografts from memory T-cell allo-responses when combined with anti-CD40L/anti-LFA-1 antibodies therapy.

ß-catenin regulates myocardial ischemia/reperfusion injury following heterotopic heart transplantation in mice by modulating PTEN pathways.

Ischemia reperfusion (I/R) injury, an inevitable event accompanying heart transplantation, is the primary factor leading to organ failure and graft rejection. In order to prevent I/R injury, we established murine heart transplantation model with I/R and cell culture system to determine whether ß-catenin is a mediate factor in preventing I/R injury in heart transplantation. After successfully established heterotopic heart transplantation mice model, the I/R injury was induced, and two dynamic temporal were studied during different I/R phases. With the increase of ischemia and reperfusion time, heart damage was more severe. In the initial study, we observed that ß-catenin was significantly decreased, while ROCK1 and PTEN increased during the perfusion phase from day 0 to day 1, and remain the same level until 3 days later. The similar pattern that ß-catenin was down-regulated while ROCK1 and PTEN were up-regulated was also observed in the dynamic temporal ischemia study. To further investigate the role of ß-catenin signaling in I/R injury in vitro, ß-catenin over-expressing plasmid was transfected into HL-1 cells, a cardiac cell line. We noted that ß-catenin over-expressing cardiomyocytes showed decreased ROCK1/PTEN expression both at mRNA and protein levels. In addition, cobalt dichloride (CoCl2) -induced oxidative stress model was further established to mimic cardiac I/R injury. We observed that CoCl2-induced activation of ROCK1/PTEN signaling pathway were attenuated by transient transfection of a ß-catenin over-expressing plasmid. Taken together, our results suggest that cardiac transplant induced IR injury is closely associated with the down-regulation of ß-catenin and up-regulation of ROCK1 and PTEN expression.

Optimization of the Cuff Technique for Murine Heart Transplantation.

Murine cardiac transplantation has been performed for more than 40 years. With advancements in microsurgery, certain new techniques have been used to improve surgical efficiency. In our lab, we have optimized the cuff technique with two major steps. First, we used the inner tube technique to insert a temporary inner tube into the external jugular vein and carotid artery blood vessel to facilitate eversion of the vessel over the cuff. Second, we performed complete heterotopic cardiac transplantation through the collaboration of two experienced surgeons. These modifications effectively reduced the operation time to 25 minutes, with a success rate of 95%. In this report, we describe these procedures in detail and provide a supplemental video. We believe that this report on the improved cuff technique will offer practical guidance for murine heterotopic heart transplantation and will enhance the utility of this mouse model for basic research.

Live birth after cervical ectopic uterus transplantation in mice.

An ideal animal model is a prerequisite for the basic research of uterus transplantation. This study aimed to develop a new cervical ectopic uterus transplantation mice model, which was established by vascular anastomosis of the right common iliac artery and vein of the donor with the right common carotid artery and external jugular vein of the recipient, respectively, using the cuff method. The survival status of the transplanted uterus was assessed by macroscopic observation and histological examination after surgery, and the function of the graft uterus was tested by verifying whether the pregnancy is possible. A total of 40 transplants were performed, of which only 1 failed due to donor hemorrhage. After 26 transplants, the total operation time reduced to 52.4 ± 3.8 minutes, of which the total ischemia time took 6.6 ± 1.1 minutes. Sixty days after transplantation, all the graft uteri had a good blood supply and spontaneous contraction. The histology showed no significant difference between the transplanted uterus and the native. Embryo transfer experiments have proven that the transplanted uterus has uterine function. In conclusion, this new model is an effective and simple mice model for the studies of the scientific issues related to uterus transplantation.

Combined treatment with vitamin D3 and antibody agents suppresses secondary heart transplant rejection in the early postoperative period.

BACKGROUND: Accelerated transplant rejection mediated by donor reactive memory T cells is another barrier to the induction of graft tolerance. The aim of this study was to investigate the immunosuppressing effects of vitamin D (1,25(OH)2D3), administered alone or in combination with a costimulatory blockade treatment, on rejection of secondary heart allografts in a mouse model. METHODS: Circular full-thickness skin grafts from BALB/c mice were cut and grafted onto the lumbar regions of C57BL/6 mice as allo-primed recipients. Four weeks after skin grafting, the vascularized hearts from the BALB/c mice were transplanted heterotopically into the allo-primed recipients using a non-suture cuff technique. The recipients were then randomly divided into four groups and given either intraperitoneal injection of isotype, Ab, 1,25(OH)2D3, or a combination of Ab and 1,25(OH)2D3. Allograft incidence was determined by hematoxylin-eosin staining, and cytokine expression was assessed by the quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assays, and cytometric bead arrays. Spleen cells from the recipient were used to assess mixed lymphocyte reactions. Memory T cells and regulatory T cells (Tregs) in spleen cells were measured by flow cytometry. RESULTS: The median allograft survival time was longer with the combined treatment with Ab and 1,25(OH)2D3 than with no treatment or with treatment with Ab or 1,25(OH)2D3 alone. The grafts were protected from infiltration by inflammatory cells and by inhibition of interleukin 2 and interferon gamma expression. Rejection was initially suppressed in the early postoperative period by a reduction in the number of memory T cells and induction of Foxp3+ Tregs, but this effect disappeared by day 15 after transplantation upon withdrawal of the treatment. CONCLUSION: Vitamin D3 administered as an immunosuppressive agent, when combined with monoclonal antibody treatment, may protect heart grafts from memory T cell responses in a secondary heart transplant model.

Generation of bioartificial hearts using decellularized scaffolds and mixed cells.

BACKGROUND: Patients with end-stage heart failure must receive treatment to recover cardiac function, and the current primary therapy, heart transplantation, is plagued by the limited supply of donor hearts. Bioengineered artificial hearts generated by seeding of cells on decellularized scaffolds have been suggested as an alternative source for transplantation. This study aimed to develop a tissue-engineered heart with lower immunogenicity and functional similarity to a physiological heart that can be used for heart transplantation. MATERIALS AND METHODS: We used sodium dodecyl sulfate (SDS) to decellularize cardiac tissue to obtain a decellularized scaffold. Mesenchymal stem cells (MSCs) were isolated from rat bone marrow and identified by flow cytometric labeling of their surface markers. At the same time, the multi-directional differentiation of MSCs was analyzed. The MSCs, endothelial cells, and cardiomyocytes were allowed to adhere to the decellularized scaffold during perfusion, and the function of tissue-engineered heart was analyzed by immunohistochemistry and electrocardiogram. RESULTS: MSCs, isolated from rats differentiated into cardiomyocytes, were seeded along with primary rat cardiomyocytes and endothelial cells onto decellularized rat heart scaffolds. We first confirmed the pluripotency of the MSCs, performed immunostaining against cardiac markers expressed by MSC-derived cardiomyocytes, and completed surface antigen profiling of MSC-derived endothelial cells. After cell seeding and culture, we analyzed the performance of the bioartificial heart by electrocardiography but found that the bioartificial heart exhibited abnormal electrical activity. The results indicated that the tissue-engineered heart lacked some cells necessary for the conduction of electrical current, causing deficient conduction function compared to the normal heart. CONCLUSION: Our study suggests that MSCs derived from rats may be useful in the generation of a bioartificial heart, although technical challenges remain with regard to generating a fully functional bioartificial heart.

Immunogenicity analysis of decellularized cardiac scaffolds after transplantation into rats.

Aim: Cardiac extracellular matrix (cECM) scaffolds are promising biomaterials for clinical applications. Our aim is to determine the immunogenicity of decellularized scaffolds from different sources for use as artificial organs during organ transplantation. Materials & methods: We transplanted Lewis rats with syngeneic (Lewis rat cECM), allogeneic (BN rat cECM) or xenogeneic (hamster cECM) decellularized cardiac scaffolds. Acute vascular and cellular rejection was quantified by immunohistochemistry and immune cell infiltration. Results: BN rat and hamster hearts were rejected following transplantation. BN and hamster cECMs had similarly low immunogenicity compared with Lewis rat cECMs and did not lead to increased rejection. Conclusion: We found that scaffolds from all sources did not induce vascular or cellular rejection and exhibited low immunogenicity.

A highly reproducible cervical cuff technique for rat-to-mouse heterotopic heart xenotransplantation.

Xenotransplantation is an effective way to solve the problem of donor shortage in clinical transplantation. However, clinical use of xenotransplantation is currently limited due to immunological challenges such as acute vascular rejection and cell-mediated rejection. To finally surpass this immunological barrier, more preclinical research is needed into the molecular mechanisms of rejection and the possible effects of new immunosuppressants. Our aim was to create a refined, highly reproducible protocol to establish the most suitable rat-to-mouse heterotopic heart transplantation model using the cuff technique.

Thoracoscopic anatomic pulmonary segmentectomy: a 3-dimensional guided imaging system for lung operations.

OBJECTIVES: To evaluate the Lung Operation Three-dimensional Imaging System software by Xiamen TRONG Technology Co. Ltd in detecting the precise position of solitary pulmonary nodules or ground-glass opacity nodules before surgery. METHODS: Chest, arterial phase, portal venous phase, enhanced and delayed-phase images of 10 cases with 12 nodules were obtained with a Toshiba Aquilion One 320 computed tomography (CT) scanner. According to the image data of the nodules, 0.5 mm thick images of 320-row multislice CT scanners were reconstructed as 3D images, and the boundary of each segment was automatically partitioned and pigmented in accordance with the pulmonary artery system. To locate the nodules, the 3D images and coloured segments were merged. The clearly labelled lung structure was utilized in a preoperative virtual segmentectomy and the subsequently planned thoracoscopic surgery. RESULTS: In all 10 cases, the reconstruction of the pulmonary artery could image branches as far as Grade 5. The anatomically adjacent relationship of the nodules among the arteries, veins and bronchi in the target pulmonary segments could be displayed in any view. The thoracoscopic anatomic pulmonary segmentectomy was performed successfully. All 12 nodules, which had deeply settled in the parenchyma, were resected using the virtual planning software and diagnosed pathologically. CONCLUSIONS: The software can be used preoperatively as a tracing method to identify the location of nodules in most general thoracic surgeries, subsequently providing guidance during the surgery.

Anti-CD44 monoclonal antibody inhibits heart transplant rejection mediated by alloantigen-primed CD4(+) memory T cells in nude mice.

Donor-reactive CD4(+)memory T cells threaten the survival of transplanted organs. In this study, we used anti-CD44 monoclonal antibody (mAb) to inhibit adoptively transferred B6-reactive CD4(+)memory T cells (BALB/c origin) and to induce tolerance of B6 hearts in nude mice. The median survival time (MST) of the grafts was 6 days in the isotype group, and more than 100 days in the group treated with 8 doses of anti-CD44 at four-day intervals. Histological analysis revealed that the mean rejection level was Grade 3 in the isotype group, and Grade 0 or 1 in the multi-dose anti-CD44 treatment group. Compared with the isotype group, the multiply treated anti-CD44 group had significantly decreased IL-2 and IFN-γ expressions, while IL-10 and TGF-ß were increased in the serum and the graft. Foxp3 in the graft was also increased. These data demonstrate that alloreactive CD4(+) memory T cells mediate the destruction of allografts, and the adhesion molecule CD44 plays an important role in this course. Anti-CD44 mAb may promote the reduction of CD4(+)memory T cells and the production of regulatory T cells (Tregs). Furthermore, Tregs are maintained at a certain level while suppressing cellular immunity and inducing the grafts long-term survival in transplant recipients.

Suppressing memory T cell activation induces islet allograft tolerance in alloantigen-primed mice.

Memory T cells are known to play a key role in prevention of allograft tolerance in alloantigen-primed mice. Here, we used an adoptively transferred memory T cell model and an alloantigen-primed model to evaluate the abilities of different combinations of monoclonal antibodies (mAb) to block key signaling pathways involved in activation of effector and memory T cells. In the adoptively transferred model, the use of anti-CD134L mAb effectively prevented activation of CD4(+) memory T cells and significantly prolonged islet survival, similar to the action of anti-CD122 mAb to CD8(+) memory T cells. In the alloantigen-primed model, use of anti-CD134L and anti-CD122 mAbs in addition to co-stimulatory blockade with anti-CD154 and anti-LFA-1 prolonged secondary allograft survival and significantly reduced the proportion of memory T cells; meanwhile, this combination therapy increased the proportion of regulatory T cells (Tregs) in the spleen, inhibited lymphocyte infiltration in the graft, and suppressed alloresponse of recipient splenic T cells. However, we also detected high levels of alloantibodies in the serum which caused high levels of damage to the allogeneic spleen cells. Our results suggest that combination of four mAbs can significantly suppress the function of memory T cells and prolong allograft survival in alloantigen primed animals.

The recall alloresponse following retransplantation is more intense compared with the T cell memory-transfer model.

The presence of alloreactive memory T cells in recipient is a critical handicap to achieving transplantation tolerance. To make a mouse model that can as closely as possible mimic the presensitized transplant patient is important for research on this subject. Thus, we developed a novel retransplant model and compared the alloresponse in this model with that in the memory T cells-transfer model (transfer control). Mean survival time of allograft was compared between 3 groups, including blank transplant control, memory transfer control and retransplant groups. Cellular rejection activity in allografts was evaluated via HE staining of cardiac graft section. Proliferation and differentiation of the alloreactive effector T cells were assayed by in vitro mixed lymphocyte reaction and flow cytometry, respectively. Real-time quantitive RT-PCR was used to assess gene expression of cytokines and surum IFN-gamma was measured via ELISA. It showed that the median survival time of allograft in retransplant recipients was significantly shortened compared to that of transfer control, and it was the same in rejection score of graft. Moreover, proliferation and differentiation of the alloreactive effector T cells were more intensive in retransplant recipients than that in transfer control, which was confirmed by in vitro mixed lymphocyte reaction and by flow cytometry of the splenocytes for detecting CD44highCD62L- memory/effector phenotype cells. Furthermore, activation of CD4+ memory T cells is reflected by high level of surum IFN-gamma and the intensive gene expression of IFN-gamma and IL-2 at cardiac allograft in retransplant recipients. Collectively, the recall alloresponse in retransplantation is more intensive than that in a memory-transfer setting, and this retransplant model is closer to the clinic situation than the memory-transfer model in rodents.

Rapamycin or tacrolimus alone fails to resist cardiac allograft accelerated rejection mediated by alloreactive CD4(+) memory T cells in mice.

Donor-reactive memory T (Tm) cells undermine transplanted organs more readily than naive T cells. Rapamycin (RAPA) and tacrolimus (FK-506) are current mainstay immunosuppressants used for preventing acute allograft rejection. Although their efficacy in suppressing naive T cell is established, their suppressing effect on memory T cells is undefined. This study was conducted to investigate the inhibiting capability of RAPA or FK-506 against transferred alloreactive CD4(+) Tm cells in a mouse cardiac transplant model. We found that these drugs alone prolonged the median survival time (MST) of allograft from 5days to 9days in recipient mice with CD4(+) Tm infusion (P<0.01), which however was not significantly longer than that (8days) in untreated recipient mice without CD4(+) Tm infusion (naive control). Mean histologic rank of rejection activity in section of cardiac allograft on day 5 postgrafting was Grade 4 in the Tm control recipients versus Grade 3A in both of the immunosuppressant treatment recipients with CD4(+) Tm infusion. RAPA or FK-506 alone failed to completely suppress proliferation and differentiation of the alloreactive CD4(+) Tm, which was confirmed by in vitro mixed lymphocyte reaction (MLR) and by flow cytometry (FCM) of the splenocytes for detecting CD44(high)CD62L(-) effector/memory as well as CD69(+)/CD25(+) activation phenotype cells from the respective recipients. Furthermore, the agent alone didn't completely inhibit the activation of CD4(+) Tm, for serum level of IFN-gamma and its gene expression at the cardiac allograft from the immunosuppressant-treated recipients were as still high as the untreated naive control. Thus, RAPA or FK-506 alone couldn't completely suppress the proliferation and activation of the alloantigen-primed CD4(+) Tm cells responding to the alloantigen, indicating that alloreactive CD4(+) Tm was insensitive to these immunosuppressants. The characteristics of alloreactive CD4(+) Tm to resist immunosuppressants and its potency to initiate quick and vigorous rejection despite treatment with the immunosuppressant make it to be a critical barrier to prolongation of allograft survival and induction of transplant tolerance.

Combined costimulation blockade inhibits accelerated rejection mediated by alloantigen-primed memory T cells in mice.

Donor-reactive memory T cells threaten the survival of transplanted organs via multiple pathways. This study was undertaken to induce tolerance of cardiac allografts in mice, in which alloreactive memory T cells were adoptively transferred, by combined costimulatory blockade of both effector and memory T cells. We found that the median survival time (MST) of the grafts was 5.17 days in the untreated group, 10.33 days in the CTLA4Ig- and anti-CD40L-treated (2-combined) group, and more than 100 days in the CTLA4Ig-, anti-CD40L-, anti-LFA-1-, and anti-OX40L-treated (4-combined) group. Histological analysis revealed that the mean rejection level was Grade 4 in the untreated group, Grade 3 in the 2-combined treatment group, and Grade 0 in the 4-combined treatment group. CD44(high) T cells were detected only in the untreated group. The in vitro proliferation of lymphocytes of both untreated and 2-combined group was higher than that of the 4-combined treatment group (p < 0.01). Compared with the untreated group, the expression levels of IL-2, IFN-gamma, and Foxp3 were lower in the 2-combined treatment group; the expression levels of these genes were the lowest in the 4-combined treatment group. IL-10 expression was significantly higher in the 4-combined treatment group than in the other groups. These results demonstrate the inhibition efficacy of combined costimulation blockade in accelerated-rejection models and the possible mechanisms underlying the suppression of cellular immunity in mice receiving grafts as well as in inducing the activation of IL-10-producing Tr1 cells in grafts.

Synergistic effects of Isatis tinctoria L. and tacrolimus in the prevention of acute heart rejection in mice.

Although immunosuppressive treatments are available for acute cardiac rejection no viable treatment exists for long-term cardiac graft failure. Moreover, the extended use of calcineurin inhibitor immunosuppressants, the mainstay of current treatment for cardiac transplantation, leads to significant side effects such as nephrotoxicity and an increased risk of cardiac disease. Because some agents used in Traditional Chinese Medicine (TCM) have strong immunosuppressive effects coupled with low toxicity, we investigated the effect of Compound K (K), the synthesized analogue of highly unsaturated fatty acids from Isatis tinctoria L., either as a single treatment or combined with tacrolimus (FK-506) on acute cardiac allograft rejection. We compared the ability of K alone, or in combination with FK-506, to inhibit acute heart transplant rejection both in vitro and in vivo. We found that the inhibition of lymphocyte proliferation was positively correlated with K concentration. K significantly reduced IL-2 and IFN-gamma expression levels and significantly inhibited lymphocyte proliferation in both a lymphocyte transformation test and a mixed lymphocyte reaction (MLR). We also found that the inhibitory effect of a combination of K and a sub-therapeutic dose of FK-506 (SubFK-506) was stronger than that of full-dose FK-506 alone. Oral administration of K reduced acute cardiac allograft rejection in mice and had no apparent toxicity. In vivo, the immunosuppressive effect of K combined with a half-dose of FK-506 was equivalent to that of a full-dose of FK-506 alone. K combined with a half-dose of FK-506 reduced the expression levels of IL-2 and IFN-gamma (both within the graft and in the recipients' serum) more effectively than a full-dose of FK-506. These results show that K has significant immunosuppressive effects both in vitro and in vivo. When used as a combination therapy with FK-506 we see a powerful inhibition of rejection with no obvious toxic side effects. The mechanism of action is postulated to involve the inhibition of IL-2 and IFN-gamma expressions by lymphocytes, rather than the activation of Tr1 cells via the production of IL-10.