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Center for the Study of Liver Disease and Department of Surgery, University of Hong Kong, Hong Kong, China
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Abstract |
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B and heat shock protein 90. In conclusion, the present study showed that the up-regulated expression of VEGF and its interaction with Flk-1 in small-for-size liver grafts might facilitate the activities of macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Similar to a liver remnant after partial hepatectomy, the transplanted small-for-size liver grafts also undergo regeneration after reperfusion, during which angiogenesis plays an essential role. Vascular endothelial growth factor (VEGF)3 secreted by replicating hepatocytes is a key player in the process of angiogenesis (6, 7), in which its major effects are on endothelial cell division and migration (8, 9). However, VEGF is also found to be a chemotaxic mediator of human monocytes (10), and a recent study even revealed that VEGF possessed proinflammatory properties that attracted leukocyte activation (11). Some other studies demonstrated that VEGF could enhance cardiac allograft arteriosclerosis (12), and might be used as a surveillance marker for cellular rejection in cardiac transplantation (13). Importantly, the effects of VEGF on its target cells are closely related to its interaction with VEGF receptors, among which VEGF-R2 (fetal liver kinase (Flk)-1) mediates most of the biological functions of VEGF. However, whether VEGF and Flk-1 in the regenerating small-for-size liver grafts, especially in the allogeneic setting, participates in mediating inflammatory and acute rejection responses remains to be determined. Therefore, in this study, we tried to elucidate the expression of VEGF and Flk-1 in liver grafts, and explored the potential role of VEGF-Flk-1 interaction in mediating the activities of macrophages.
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Materials and Methods |
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Orthotopic liver transplantation (14) was performed using adult male Dark Agouti (RT1a) and Lewis (RT1l) rats as donors and recipients, respectively. The percentage of grafts was determined according to experimental protocols. A total of six experimental groups were included: 1) whole isograft (Lewis to Lewis), n = 9; 2) 50% isograft, n = 9; 3) 30% isograft, n = 9; 4) whole allograft (Dark Agouti to Lewis), n = 12; 5) 50% allograft, n = 12; and 6) 30% allograft, n = 12. Both 50 and 30% grafts were regarded as small-for-size grafts. No treatment was given to the groups. Animals were sacrificed at 6, 24, and 72 h after reperfusion (3–4 animals at each time point in each group) for tissue and plasma collection.
RT-PCR, Western blot, and ELISA
Total RNA extraction, first-strand DNA synthesis, and PCR were performed according to the methods previously described (5). Primers forVEGF were designed according to Liu et al. (15). Primer sequences for rat Flk-1 were: sense, 5'-CCAAGCTCAGCACACAAAAA-3' and antisense, 5'-CCAACCACTCTGGGAACTGT-3'. Protein levels in tissue were determined by standard Western blot analysis using 12% SDS-PAGE gel (monoclonal mouse anti-rat VEGF and polyclonal rabbit anti-rat Flk-1 Abs were purchased from Santa Cruz Biotechnology, Santa Cruz, CA). Soluble VEGF levels in plasma were detected by ELISA according to the protocol suggested by the manufacturer (R&D Systems, Minneapolis, MN).
Cell culture
Cell lines.
Macrophage cell lines CRL-2192 were purchased from the American Type Culture Collection (Manassas, VA). Cells were maintained in F-12K medium with 15% FBS and 1% penicillin-streptomycin (Invitrogen Life Technologies, Carlsbad, CA) at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were cultured in serum-free medium for 12 h before different doses of rat VEGF (R&D Systems), 5 µg/ml Flk-1 inhibitor (E)-3-(3,5-diisopropyl-4-hydroxyphenyl)-2-[(3-phenyl-n-propyl)amino-carbonyl]acrylonitrile (SU1498; Calbiochem-Novabiochem, San Diego, CA), 10 ng/ml rat IL-1
, 10 ng/ml TNF-
(BioVision, Mountain View, CA), 400 µM desferrioxamine (Sigma-Aldrich, St. Louis, MO), 2 mM sodium salicylate (Santa Cruz Biotechnology), and 1 µg/ml heat shock protein 90 (Hsp90) inhibitor, geldanamycin (Sigma-Aldrich) for 12 h according to treatment regimens.
Primary culture. Mononuclear cells were isolated from the peripheral blood of recipients at 72 h after reperfusion using Ficoll gradient centrifugation. The isolated mononuclear cells (1 x 106) were then treated with 5 ng/ml VEGF, 5 µg/ml SU1498 (Flk-1 inhibitor), and VEGF combined with SU1498, respectively, for 12 h before flow cytometry analysis. Mononuclear cells were also isolated from liver grafts according to the method described by Curry et al. (16) for phenotypic analysis by flow cytometry.
Flow cytometry
Cells from the primary culture were labeled with FITC-conjugated mouse anti-rat ED1 and PE-conjugated mouse anti-rat CD80 or CD86 mAbs (Serotec, Oxford, U.K.), or PE-conjugated mouse anti-rat ED1 (Serotec) and FITC-conjugated rabbit anti-rat Flk-1 Abs (Santa Cruz Biotechnology), and detected in FACSCalibur (BD Immunocytometry Systems, San Jose, CA). Cells from macrophage cell lines were labeled with FITC-conjugated rabbit anti-rat Flk-1 Ab (Santa Cruz Biotechnology) and also detected in FACSCalibur (BD Immunocytometry Systems). Appropriate isotypes of irrelevant Abs were used as controls.
Cell migration assay
Cells from macrophage cell lines were suspended in 50 µl F-12K medium with 1.5% FBS and inoculated on the surface of a polycarbonate filter of a 96-well microchemotaxis chamber (1 x 105 per well; NeuroProbe, Bethesda, MD). In the lower chamber, 299 µl of 15% FBS-F-12K medium, or 1.5% FBS-F-12K medium with either 5 ng/ml VEGF, 5 µg/ml SU1498, or VEGF combined with SU1498 was applied into each well, respectively. The cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in air for 4 h before they were counted under microscope. Five areas were randomly chosen in each well and the number of cells was expressed as mean ± SD.
Statistical analysis
Comparisons of plasma VEGF levels, the number of Flk-1+ cells in macrophage cell lines or double-positive cells in primary culture between different treatment groups were performed using Student’s t test (GraphPad, San Diego, CA). Values of p < 0.05 were considered as statistically significant.
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Results |
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At 6 h after reperfusion, up-regulation of VEGF at both mRNA and protein levels were detected in the small-for-size graft groups in both syngeneic and allogeneic combinations, particularly in 30% grafts (Figs. 1, A and B, and 2, A and B). In both 30% isograft and allograft groups, VEGF levels in plasma detected at 6, 24, and 72 h after reperfusion were significantly higher than whole isograft and allograft groups, whereas the increased plasma levels of VEGF in 50% isograft and allograft groups were detected at 6 and 24 h after reperfusion (Figs. 1C and 2C). In addition, the mRNA and protein levels of Flk-1 were also increased in the small-for-size liver grafts at 6, 24, and 72 h after reperfusion in both isogeneic and allogeneic settings (Figs. 1, D and E, and 2, D and E). At 72 h after reperfusion, an increased number of Flk-1+ED1+ cells were detected in the small-for-size isograft and allograft groups, compared with whole isograft and allograft groups (Fig. 3).
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By RT-PCR and Western blot, it was found that the macrophage cell lines did not initially express detectable Flk-1 mRNA and protein, but could be induced by desferrioxamine and, preferably, VEGF. However, the expression of Flk-1 on macrophages could not be stimulated by inflammatory cytokines (IL-1 and TNF-; Fig. 4A). The number of Flk-1+ macrophages was increased with the stimulation of desferrioxamine and, especially, VEGF (Fig. 4B), and the highest number of Flk-1+ macrophages was induced by 5 ng/ml VEGF. However, with the increased doses of VEGF (10 and 20 ng/ml), the number of Flk-1+ macrophages did not further increase, though it was still significantly higher than the no treatment group (Fig. 4C).
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To identify the role of VEGF and Flk-1 on macrophage migration, a migration assay was performed using VEGF as a chemotaxic attractor. Similar to 15% FBS, 5 ng/ml VEGF in 1.5% FBS-culture medium could induce a significantly higher number of macrophage migration than culture medium with 1.5% FBS only, whereas the migration behavior was inhibited by SU1498 (Flk-1 blocker) administration (Fig. 5).
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To further clarify the role of VEGF and Flk-1 expression on monocyte proliferation (CD80 and CD86 were used as markers to label activated monocytes), mononuclear cells were isolated from the peripheral blood of the recipients at 72 h after reperfusion, and cultured with 5 ng/ml VEGF, 5 µg/ml SU1498, and VEGF combined with SU1498, respectively, for 12 h. In both syngeneic and allogeneic combinations, VEGF could remarkably increase the number of ED1+CD80+ and ED1+CD86+ cells after a 12-h culture in both the whole and small-for-size graft groups. However, the increase of ED1+CD80+ and ED1+CD86+ cells was reduced when SU1498 was administered with VEGF in all the small-for-size graft groups and the whole allograft group, but not the whole isograft group (Figs. 6 and 7).
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B and Hsp90
To explore the possible molecules that might be related to Flk-1 expression on macrophages, the nonsteroid anti-inflammatory drug, sodium salicylate, and the Hsp90 inhibitor, geldanamycin, were introduced to macrophage cell lines with VEGF. It was found that VEGF could augment the NF-B (p65) protein level, but it had no obvious effect on Hsp90. However, administration of sodium salicylate with VEGF could reduce the levels of both p65 and Hsp90. Similarly, administration of geldanamycin to VEGF could also decrease the level of Hsp90 and reverse the up-regulated p65 level induced by VEGF (Fig. 8, A and B). At the same time, the increased number of Flk-1+ cells induced by VEGF was completely reversed by sodium salicylate, and partially by geldanamycin (Fig. 8C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The interaction between VEGF and its receptors plays a crucial role in mediating monocyte and macrophage activities. It was reported by Sawano et al. (10) that human monocytes only expressed VEGF-R1 (fms-like tyrosine kinase-1), but not Flk-1. However, in the situation of small-for-size liver transplantation, we did find a group of cells that expressed both macrophage marker-ED1 and Flk-1 in both isografts and allografts, suggesting that macrophages might express Flk-1 in certain circumstances. However, we did not detect Flk-1 expression in macrophage cell lines when no treatment was given. When the cells were stimulated by iron chelator desferrioxamine (desferrioxamine could induce endogenous VEGF production in various types of cells through generating a hypoxic condition; Refs. 21 and 22) and exogenous VEGF, an increased expression of Flk-1 was detected preferably by the exogenous VEGF. Additionally, the inducible expression of Flk-1 on macrophages by VEGF displayed a dose-dependent manner and could reach a peak level with the dose of 5 ng/ml, implying that Flk-1 might be an inducible receptor on macrophages, and exogenous VEGF stimulation might function more effectively on Flk-1 expression. However, we did not observe any noticeable Flk-1 changes in the liver grafts at different time points after reperfusion, during which an increased expression of VEGF was detected. This might be because macrophages were not the only cell population that expressed Flk-1, and more abundant Flk-1 expression was found to be expressed in endothelial cells (23) and hepatic stellate cells (21). Whether the expression of Flk-1 in these types of cells can be augmented by VEGF remains to be determined.
The processes of cell migration and proliferation were two key steps of macrophage activation. It was reported that VEGF could induce monocyte migration in an fms-like tyrosine kinase-1-dependent manner (10). The present study further demonstrated that macrophage migration mediated by VEGF might also depend on Flk-1 expression. This was supported by the evidence that the number of migrated cells by VEGF could be partially reversed with Flk-1 inhibition. However, the question of which of the two VEGF receptors plays a more predominant role needs to be determined. In addition, the expression of Flk-1 on monocytes also contributed to the proliferation of activated monocytes isolated from the peripheral blood of liver graft recipients, as the increased number of CD80+ and CD86+ monocytes by VEGF could be partially reversed by Flk-1 blockade, indicating that VEGF-stimulated monocyte proliferation was partially dependent on Flk-1 expression. The only exception was in the whole syngeneic transplantation. Although we could also observe an increased number of CD80+ and CD86+ monocytes after VEGF stimulation, the augmentation was not inhibited by Flk-1 blockade, indicating that the number of activated monocytes at the time of VEGF stimulation might also be important for the effects of Flk-1. In other words, the effects of VEGF-Flk-1 interaction on monocyte proliferation required a certain number of activated cells. However, further studies are needed to clarify the potential mechanisms. Because several other factors (e.g., proinflammatory cytokines) might also contribute to the activation of monocytes and macrophages, and a significantly increased expression level of VEGF in allografts was detected only after 6 h, it further suggested that the most important role of VEGF on macrophages and monocytes was enhancement of their migration and proliferation activities rather than initiation.
Finally, we demonstrated in the present study that up-regulation of Flk-1 on macrophages by VEGF was associated with the expression of NF-B (p65) and Hsp90. It has been reported that VEGF could activate NF-B in a cell type-dependent manner (24), and the activity of NF-B was related to the activation of the Flk-1 promoter (25). Therefore, introduction of VEGF to macrophages might induce the activation of Flk-1. Our study revealed that the up-regulation of Flk-1 on macrophages by VEGF could be reversed by administration of sodium salicylate. At the same time, a down-regulation of p65 was detected in these cells, further suggesting that the activation of Flk-1 was associated with the activity of NF-B. Hsp90 was found to be crucial in determining leukemia cell survival induced by VEGF (26). In addition, Hsp90 was an important chaperone of several molecules including the tyrosine kinase family, which played crucial roles in the signal transduction cascade that mediated inflammatory activities of mast cells (27). Hsp90 was also found to be able to enhance the activities of inducible nitric-oxide synthase (28) and cytokine production of macrophages (29). Our study suggested that the induction of Flk-1 activation by VEGF was also related to Hsp90, as the increased number of Flk-1+ macrophages could be partially reduced by the Hsp90 inhibitor, geldanamycin. Further studies are needed to explore the potential mechanism.
In conclusion, our study revealed that VEGF production and VEGF receptor expression were increased in the small-for-size liver grafts during the early period after reperfusion, and up-regulated VEGF expression might enhance monocyte and macrophage activities in a VEGF-R2 (Flk-1)-dependent manner.
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Footnotes |
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1 This study was supported by a research grant from the Distinguished Research Achievement Award and Sun Chieh Yeh Research Foundation for Hepatobiliary and Pancreatic Surgery of the University of Hong Kong. 
2 Address correspondence and reprint requests to Dr. Sheung Tat Fan, Department of Surgery, University of Hong Kong Medical Centre, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong, China. E-mail address: hrmsfst{at}hkucc.hku.hk
3 Abbreviations used in this paper: VEGF, vascular endothelial growth factor; Hsp90, heat shock protein 90; Flk, fetal liver kinase; SU1498, (E)-3-(3,5-diisopropyl-4-hydroxyphenyl)-2-[(3-phenyl-n-propyl)amino-carbonyl]acrylonitrile.
Received for publication March 1, 2004. Accepted for publication June 8, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in renal cell carcinoma associated with loss of Tsc-2 tumor suppressor gene. Cancer Res. 63:2675.B activation in hematopoietic progenitor cells. Cancer Res. 61:2015.B and cAMP response element binding protein mediate opposite transcriptional effects on the Flk-1/KDR gene promoter. Circ. Res. 86:E110.
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, p < 0.05, compared with the number of cells isolated from the whole isograft group; *, p < 0.05, compared with the cell number without treatment (cells from the whole isograft group); #, p < 0.05, compared with the cell number without treatment (cells from the 50% isograft group); 
, p < 0.05, compared with the cell number without treatment (cells from the 30% isograft group); ##, p < 0.05, compared with the cell number after VEGF culture (cells from the 50% isograft group); 
