Background. Vascular endothelial growth factor (VEGF), a major angiogenic factor, mediates a variety of disease conditions through promotion of angiogenesis. It also plays a critical role as a potent proinflammatory cytokine in a variety of physiologic and pathologic immune responses. In the present study, we evaluated the expression of VEGF in hepatic warm ischemia-reperfusion (I/R) injury and examined the effect of recombinant human (rh)VEGF administration in an established murine model. Method. The expression of VEGF in the liver was assessed by quantitative real-time polymerase chain reaction and immunohistochemistry during I/R injury using 70% partial hepatic ischemia model. The effect of rhVEGF administration on I/R injury was evaluated by measuring liver function and histology. In addition, local inducible nitric oxide synthase (iNOS) and endothelial NO synthase expressions were examined to address the underlying mechanisms. Results. The local expression of VEGF was significantly up-regulated at 2 hours after reperfusion after 60 minutes of ischemia compared with that in the naïve liver. VEGF was expressed predominantly in CD11b+ cells infiltrating into the ischemic liver. The administration of rhVEGF had a significant protective effect on ischemic injury in the liver. This effect was associated with the up-regulation of iNOS expression in the rhVEGF-treated liver. Conclusion. We demonstrate a dual role of VEGF in hepatic warm I/R injury. Although endogenous VEGF is expressed and functional to initiate hepatic I/R injury, exogenous rhVEGF has a beneficial effect on the ischemic liver. These data may provide new insights into the role of VEGF as well as pathophysiology of hepatic I/R injury.
O183* Aims: Vascular endothelial growth factor (VEGF), a major angiogenesis factor, is also a pro-inflammatory cytokine and plays a critical role in a variety of physiological and pathological immune response. It is well established to be induced by hypoxia, suggesting that its expression will be characteristic of transplantation. However, little is known of its role in post-transplantation ischemia/reperfusion (I/R) injury. In this study, we investigated the expression and function of VEGF and its receptors (flt-1 and flk-1) in I/R injury. Methods: We used a murine hepatic I/R injury model. Seventy percent partial hepatic ischemia was performed for 60 or 120 minutes in male C57BL/6 mice. To address the underlying mechanisms, we utilized quantative real-time PCR and histological analysis. Results: First, we evaluated the local expression of VEGF and receptors in the liver using quantative real-time PCR in the process of hepatic I/R injury. VEGF expression was significantly up-regulated after 2h of reperfusion following 60 minutes of ischemia comapred to that in the naive liver (n=4, p=0.021). In addition, flk-1 expresson was significantly down-regulated (n=6, p=0.006). Data suggested that VEGF and the receptors might play an important role in the initiaion of hepatic I/R injury. Next, we administrated anti-flk-1 (DC101) and anti-flt-1 (MF-1) neutralizing monoclonal antibodies (mAb, 1mg of each) 30 minutes before reperfusion. Interestingly, mAb treatment significantly inhibited hepatic injury compared to control at 6 h of reperfusion (n=8 of each, sAST: 2047±317 vs. 5404±1078: p=0.0098, sALT:3637±577 vs. 10590±2060: p=0.0058, sLDH:7635±1112 vs. 23362±5059: p=0.0089). Histological analysis also revealed the protective effect of targeting VEGF receptors on hepatic damage. Massive cellular infiltration and extensive hepatic cellular necrosis was observed in control mice, while the lobular architecture was relatively preserved and there was less necrosis in mice treated with mAb of VEGF receptors. Since VEGF may function in vivo via alterations in leukocyte trafficking, we also evaluated intragraft expression of cytokines and adhesion molecule in control and antibody-treated liver using quantative real-time PCR. We found that blockade of flt-1 and flk-1 significantly down-regulated the local expression of several cytokines (TNF-α, IFN-γ and IL-10, p=0.004, p=0.003 and p=0.004, respectively vs control) and the adhesion molecules (E-selectin and ICAM-1, p=0.0005 and p=0.0042) after 2h of reperfusion following 60 minutes of ischemia. Finally, the removal of shunt liver after 120 minutes of ischemia resulted in the death of all mice treated with saline within 7 days. By sharp contrast, 40% of mice treated with anti-VEGF receptor antibodies survived indefinitely. Conclusions: This study demonstrates for the first time that VEGF is expressed and is functional in hepatic I/R injury. We suggest that blockade of VEGF via inhibition of its receptors may represent a novel target for the protection of the liver in the peri-transplant period.
Background. We have recently reported that vascular endothelial growth factor (VEGF) functions as a proinflammatory cytokine to regulate the trafficking of leukocytes into allografts in the early posttransplant period. VEGF binds two major VEGF receptors: VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR). Here, we wished to investigate the expression and function of VEGF receptors in the process of acute allograft rejection in vivo. Methods. We performed fully MHC-mismatched C57BL/6 (H-2b) into BALB/c (H-2d) vascularized heterotopic murine cardiac transplants and we examined the expression of VEGF and VEGF receptors by immunohistochemistry during acute allograft rejection. Next, we treated mice with specific neutralizing monoclonal antibodies against murine VEGFR-1 and VEGFR-2 and examined their effect on the development of acute allograft rejection by histology and by analysis of graft survival. The intragraft expression of cytokines and chemokines were also evaluated by quantitative real-time PCR analysis. Results. The expression of VEGF, VEGFR-1 and VEGFR-2 were significantly up-regulated during allograft rejection as compared to isografts. Administration of either anti-VEGFR-1 or anti-VEGFR-2 alone failed to inhibit allograft rejection. However, coadministration of both antibodies together inhibited leukocyte infiltration of allografts and prolonged allograft survival. Furthermore, the effect of VEGFR blockade was associated with the downregulation of intragraft cytokine and chemokine expression. Conclusions. Our data suggest that VEGF-VEGFR interactions function in the alloimmune response in vivo. Targeting VEGFRs may represent a novel therapy to protect allografts following clinical transplantation.
A 67-year-old man was referred to the department with a diagnosis of esophageal cancer. On admission, a decrease in platelet count, 6.2×10/m3 and a slight increase in PAIgG were noted. So the patient was diagnosed as having idopathic thrombocytopenic purpura (ITP) as well. The patient underwent high-dose γ-globulin therapy (286mg/kg/day) for 5 consecutive days prior to surgery. After confirming an increase in platelet count from 6.2×104 to 16.7×104/mm3, the patient safely underwent a subtotal esophagectomy, regional lymph node dissection (R3) and splenectomy. Although anastomotic insufficiency occurred, the complication was cured without surgical procedure. We believe that the high-dose γ-globulin therapy that is able to increase platelets in a short time and has minimal adverse effects is very useful in preoperative management of the patients with esophageal cancer with ITP.
O387* Aims: We have recently reported that vascular endothelial growth factor (VEGF), a major angiogenesis factor is functional as a proinflammatory cytokine in acute allograft rejection (J. Clin. Invest. 112, 2003). In this study, we further investigated the role of VEGF receptors and anti-angiogenic factor in alloimmune response in vivo. Methods: We used two distinct murine cardiac transplant models. In one model, C57BL/6 hearts were transplanted into fully MHC mismatched BALB/c recipients. In the other model, B6.C-H2bm12 hearts were transplanted into MHC class II disparate C57BL/6 recipients to evaluate the progressive graft vascular disease (GVD). Results: We first investigated the importance of two high affinity receptors for VEGF: flt-1 and flk-1. Treatment of anti-flt-1 (MF1) and anti-flk-1 (DC101) monoclonal antibodies significantly prolonged cardiac allograft survival (MST=16.0 days, P=0.004 vs. control; MST=8.0 days). Data suggested that VEGF might function via these two receptors in the process of acute allograft rejection. Next, we evaluated the therapeutic efficacy of murine endostatin/Fc, a potent anti-angiogenic factor in alloimmune response. We found that murine endostatin/Fc treatment significantly prolonged allograft survival in a dose-dependent manner (20 mg/kg, 0-7 days; MST=10 days, 40 mg/kg; MST=36.7 days, p=0.041 and p=0.0002, respectively, vs. control). To address the underlying mechanisms, we performed quantative real-time PCR analysis. Although the effect of endostatin/Fc was not associated with neovascularization in this acute model, the treatment significantly downregulated the local expression of cytokines (IFN-γ and IL-4, p=0.0416 and p=0.005 vs. control), chemokine (RANTES, p=0.0249) and chemokine receptor (CXCR3, p=0.007). Furthermore, there was seen less infiltration of CD4 and CD8 T cells (p=0.001 and p=0.043 vs. control) into cardiac allografts. Data suggested that endostatin/Fc inhibited acute alloimmune response through downregulation of local immune activation. Finally, we evaluated the effect of endostatin/Fc on the development of GVD as a hallmark of chronic rejection. C57BL/6 recipients that were transplanted with B6.C-H2bm12 cardiac allografts were treated with endostatin/Fc (20mg/kg three times a week), either in the initiation phase (0-3 weeks) or in the progression phase (4-6 weeks). Remarkably we found that early treatment failed but that delayed treatment significantly inhibited the development of GVD (n=8 of each, P=0.026 vs. control). Moreover, there was less fibrosis and less cellular infiltration of grafts in the delayed treatment group. Conclusions: Our data provide important new insights into the significance of angiogenic factor in alloimmune response. Furthermore, this study demonstrates for the first time that a potent anti-angiogenic factor, endostatin can be therapeutically effective in the process of chronic as well as acute allograft rejection. We suggest that angiognesis may represent a novel target for the pretection of alloimmune response in clinical transplantation.
