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A novel recombinant fibrinogenase of Agkistrodon acutus venom protects against hyperacute rejection via degradation of complements
Biochemical Pharmacology 85 (2013) 772–779
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A novel recombinant fibrinogenase of Agkistrodon acutus venom protects against hyperacute rejection via degradation of complements Xi Lin a,1,*, Jie-zhen Qi b,1, Ming-hui Chen c, Bi-tao Qiu d, Zhen-hua Huang b, Peng-xin Qiu b, Jia-shu Chen b,**, Guang-mei Yan b,*** a
Department of Pharmacology, Medical College, Ji-Nan University, 601 Huangpu Road, Guangzhou, Guangdong 510632, PR China Department of Pharmacology, Zhong-Shan Medical College, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou, Guangdong 510089, PR China Department of Gynecology and Obstetrics, First Affiliated Hospital of Sun Yai-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080, PR China d School of Biology Sun Yat-sen University, 135 Xingangxi Road, Guangzhou, Guangdong 510275, PR China b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 September 2012 Accepted 13 November 2012 Available online 23 November 2012
Hyperacute rejection (HAR) is a main barrier in xenotransplantation, which is mediated by the combination of natural antibody to the xenograft and complement activation. Current therapies have focus on the inhibition of complement by development of complement inhibitor and transgenic animal organ. Here, we investigated the effects of rFII, a recombinant fibrinogenase from Agkistrodon acutus venom, on complement and HAR. The degradation effect of rFII on complement was tested by SDS-PAGE, CH50 examination, ELISA Kit and cofocal immunofluorescence microscopy in vitro and in vivo. An exvivo rat-to-human perfusion model and a vivo guinea-pig-to-rat heat HAR model were used to determine the protection of rFII against HAR. Our investigation indicated that rFII could significantly degrade human C5, C6, and C9, decrease the activity of complement, and inhibit the MAC deposition on HUVECs membrane in vitro. In addition, serum levels of C1q, C3 and C4 in rat were gradually reduced after infusion of rFII. Importantly, in an ex vivo rat-to-human perfusion model, the survival of rat hearts perfused with human serum treated with rFII (83.36 16.63 min) were significantly longer than that of hearts perfused with fresh human serum(15.94 4.75 min). At the time of 15 minutes after perfusion, functions of hearts added with 50 ug/ml rFII sustained well with heart rates at 283 65.32 beats/minute and LVDP at 13.70 5.45 Kpa, while that of hearts perfused with fresh human serum were severely damaged by HAR with heart rates at 107.77 40.31 beats/minute and LVDP at 1.01 0.83 Kpa. We also found that rFII significantly decreased the levels of C1q, C3 and C4 in human fresh serum perfusate. In a vivo guinea-pig-torat heat HAR model, the survival of rat hearts treated with rFII were significantly longer than that of hearts perfused with normal saline; and relieved heart damage by complete activation. Our finding demonstrates the anti-complement property of rFII and its protection against HAR, indicating that rFII might be as a potential therapeutic agent for xenotransplantation. ß 2012 Elsevier Inc. All rights reserved.
Keywords: Xenotransplantation Hyperacute rejection Complement
1. Introduction
Abbreviations: ADP, adenosine diphosphate; ATII, antithrombin; CH50, complement hemolytic activity 50; CVF, cobra venom factor; HAR, hyperacute rejection; HUVEC, human umbilical vein endothelial cell; HR, heart rate; IgM, immunoglobulin M; LVDP, left ventricular developed pressure; LSCM, laser scanning confocal microscopy; MAC, membrane attack complex; rFII, recombinant fibrinogenase of Agkistrodon acutus venom; SCR1, soluble human complement receptor type1; XNAs, xenoreactive natural antibodies. * Corresponding author. Tel.: +8620 85220242; fax: +8620 85228865. ** Corresponding author. Tel.: +8620 87330553; fax: +8620 87330553. *** Corresponding author. Tel.: +8620 87330578; fax: +8620 87330578. E-mail addresses: [email protected] (X. Lin), [email protected] (J.-s. Chen), [email protected] (G.-m. Yan). 1 Co-first authors. 0006-2952/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2012.11.012
Clinical transplantation is the only effective therapy for endstage organ failure. Unfortunately, many patients do not receive this therapy owing to the severe shortage of suitable human organ donors [1]. This supply-demand imbalance could be corrected by transplanting organs from other species (xenografts) into human. The most major barrier to xenotransplantation, however, is hyperacute rejection (HAR) to the organ graft. HAR is mediated by the binding of preexisting of xenoreactive natural antibodies(XNAs) to antigens expressed on the donor organ endothelia, which active complement and finally lead to thrombosis and destruction to the graft [2,3]. The antibody and complement system have been shown to play a central pathophysiologic role in HAR and to contribute to the inflammation and
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organ injury associated with transplantation [4,5]. Furthermore, increasing evidence suggested that coagulation and thrombotic disorders were associated with HAR as well, which was characterized by the formation of platelet adhesion, aggregation, thrombosis, and endothelial dysfunction resulting from activation of the coagulation pathway [6,7]. Thus, interventions made to overcome HAR mainly concentrated on prevention of complement activation or removal xenoreactive natural antibodies [1,8] and therapeutic strategies to prevent intravascular thrombosis after xenotransplantation have received increasing attention as well. Despite these advances, development of an effective drug that has therapeutic role against HAR is still in urgent. Our previous results revealed that rFII, a novel recombinant fibrinogenase from Agkistrodon acutus venom could inhibit the platelet aggregation and directly degrade the microvascular thrombosis without activating intrinsic plasminogen [9–11]. Based on the hypothesis that inhibition of both complememnts and coagulation cascade could be a clue to avoid HAR, an exploration for the possible therapeutic potential of rFII against HAR is therefore essential. Here we demonstrated that rFII cleavaged and decreased the activity of complements in vitro, reduced the serum level of complements in vivo and protected against HAR in a rat heart ex vivo perfusion model and a guinea pig-to-rat heart HAR model, indicating that it might be an potential drug candidate against HAR. 2. Materials and methods 2.1. Reagents, cell cultures and animals The recombinant fibrinogenase II(rFII) from Agkistrodon acutus venom was prepared as we previously described [11]. Human Umbilical Vein Endothelial cells (HUVECs) were obtained from the American Type Culture Collection (Manassas, USA). Cells maintained in DMEM (Invitrogen, USA) supplemented with 10% FBS and a humidified atmosphere of 5% CO2 at 37 8C. All animal experiments were conducted in accordance with the National Guide for the Care and Use of Laboratory Animals and were approved by Sun Yat-sen University Animal Care and Use Committee (Guangzhou, China). Adult male S-D rats (250–300 g) and adult male guinea-pigs (300–350 g) were supplied by the Experimental Animal Center of Zhongshan Medical College, Sun Yat-san University. 2.1.1. Degradation effect of rFII on human complement in vitro Human complement C5, C6 and C9 (Sigma Aldrich, USA) were incubated with 10 ug/ml rFII at 37 8C for 2 h. Then samples were analysed by 12% SDS-PAGE. 2.1.2. Complement hemolytic activity (CH50) assay The complement hemolytic activity (CH50) was detected as previously described [12]. The red cells of guinea-pig were diluted to the concentration of 2% by the buffer (NaCl17g, Na2HPO4 11 g, KH2PO4 0.27 g, 10% MgCl2 2 ml and distilled water 280 ml). Then the dilutions were incubated with isovolumic hemolysin at 37 8C for 0.5 h. The SD rat serums were treated with or without rFII for different times (2 h, 6 h and 12 h). We established four groups: low-dose (10 mg/ml rFII) group, medium-dose(20 mg/ml rFII) group, high-dose(50 mg/ml rFII) group and normal group. The appropriate series of treated or untreated rat serum were added into a series of reaction tubes containing the buffer and sensitized guinea-pig red cells. After incubation at 37 8C for 1 h, the tubes were centrifuged to remove unlysed cells, and the optical density of supernatant was measured at 540 nm. The quantity of complement required for 50% hemolysis is defined as CH50 value.
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Data were expressed as the percent change in CH50 at selected time point which was calculated using the following formula: (CH50 value at the selected time point/pre-injury baseline value 100%). 2.1.3. Degradation effect of rFII on serum complement in vivo Rats were infused with rFII for 6 h at the speed of 1 ml/h through the right femoral vein. Four different groups were established: normal group: saline was infused; rFII groups: 0.1 mg/kg, 0.2 mg/kg and 0.5 mg/kg. The plasma of rats were collected in tube at pre-infusion (0 h) and post-infusion (2 h, 4 h and 6 h) time points and stored at 20 8C until assayed. The concentrations of C1q, C3 and C4 in animal plasma was determined using ELISA Kit (R&D, USA). 2.1.4. Ex-vivo rat hearts perfused with human serum supplemented with rFII This working heart–perfusion model used for hyperacute rejection was prepared as previously described [13]. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbitone (100 mg/kg). Heparin (200IU/100 g) was injected via the inferior vena cava, and the heart was quickly removed to ice-cold Krebs-Henseleit buffer (K-H buffer) containing (mM): NaCl 118, KCl 4.7, CaCl2 1.75, MgSO4 1.2, glucose 11, EDTA 0.5, NaHCO3 25. Following aortic cannulation, hearts were perfused in the ex vivo Langendorff mode at a constant flow rate of 15 ml/min with oxygenated (95%O2 and 5% CO2) perfusate at 37 8C. Two electrodes were put on the left ventricle and the right atrium to monitor heart rate (HR) and left ventricular developed pressure (LVDP). Human blood was obtained from healthy volunteers after informed consent, separated into serum and cellular components by centrifugation. The hearts of 60 male SD rats (200–300 g) were explanted. Administration was started after 30-min equilibration period. Six groups were established: serum-control group was perfused with fresh human serum and served as a positive control; decomplementation group was perfused with the decomplemented human serum which was performed by heating of the serum at 56 8C for 30 minutes to inactivate the complement activity as negative controls. rFII-treated groups (low-, medium-, and highdose rFII) were given the human serum which incubated with different doses of rFII(10 ug/ml, 20 ug/ml and 50 ug/ml) for 6 h at 37 8C before perfusion. The additional isolated hearts, which were given neither treated- nor untreated- human serum, were infused with K-H buffer. The experiments were terminated when hearts failed to pump against the afterload column of 55 mm Hg and the survival time of perfused hearts was recorded. The serum were collected in tube before and after experiments and stored at 20 8C The concentrations of C1q, C3 and C4 in human serum were determined using ELISA Kit (R&D,USA). 2.1.5. Guinea-Pig-to-Rat heart transplantation in vivo S-D rat underwent a heterotopic guinea-pig-heart transplantation into the abdomen as previously described [13]. The transplant was performed through a lower midline abdominal incision, implanting the guinea-pig heart by anastomosis of the donor aorta to the recipient abdominal aorta and of donor pulmonary artery to the recipient inferior vena cava. Treatments were started 6 h before heterotopic heart transplantation through femoral vein. Four groups were established, rFII-treated groups (low-, medium-, and high-dose rFII) were given different doses of rFII(0.1 mg/kg, 0.2 mg/kg, and 0.5 mg/kg) for 6 h. Normal group: saline was infused. Guinea-pig heart xenografts were removed at the time of rejection or death for histological study with H/E staining, and immunohistochemically for immunoglobulin C3.
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2.1.6. Confocal immunofluorescence microscopy of C5b-9 neoantigen We evaluated the effects of rFII on MAC(membrane attack complex) through assembling C5b-9 on the membranes of HUVECs. Assembly of sublytic C5b-9 on the membranes of HUVECs was done with using purified complement complex, including C5b6 (1.0 mg/ml), C7 (10 mg/ml), C8 (10 mg/ml) and C9 (10 mg/ml) according to the manufacture’s instructions (Calbiochem, USA). Four groups were established: normal control group, pre-rFII groups (1, 10 mg/ml, respectively), post-rFII group (10 mg/kg). Laser scanning confocal microscopy assay (LSCM) was used to assess C5b-9 neoantigen localization on membranes of HUVECs. Cells were fixed in a 1:1 methanol: acetone solution for 30 min following by blocking in goat serum for 1 h. 1:200 monoclonal anti-C5b-9 antibody (Invitrogen, USA) were added to the cells and incubated at 4 8C overnight. FITC-conjugated goat anti-rat IgG (Cell Signaling Technology, USA) was added for 1 h at room temperature and 1:1000 Hoechst33258 (Molecular Probes, USA) was added for 15 minutes in the dark and mounted to glass slides until visualized by confocal immunofluorescence microscopy (Nikon, Japan). 2.1.7. Histopathological Analysis Heart samples were fixed in 10% neutral-buffered formalin, embedded in paraffin, and stained with hematoxylin-eosin. 2.1.8. Immunohistochemistry Immunohistochemistry staining of 4 mm sections of paraffinembedded samples were performed as described [14]. Sections were stained with 1:200 primary rabbit anti-rat antibody C3 (invitrogen, USA) overnight at 4 8C, bio-anti rabbit IgG for 1 hour and then incubated with avidin biotin-peroxidase complex diluted in NaCl/Pi. 2.2. Statistical analysis Differences between data groups were assessed for significance using Student’s t-test of unpaired data or one-way analysis of variance. Repeated measures analysis of variance was used for multivariate analysis. All experiments were repeated at least three times and the data were presented as the mean S.D.
3. Results 3.1. Proteolytic effect of rFII on human complements in vitro To investigate whether rFII has a proteolytic effect on human complements in vitro, we tested the protein level of human complements after pre-incubation with rFII. SDS-PAGE analysis showed that the human complements(C5, C6, and C9) were dramatically degraded after pre-incubation with 10 ug/ml rFII for 2 h at 37 8C (Fig. 1). Consistently, the activity of complement was also significantly decreased as the time of pre-incubation with rFII prolonged (Table 1), suggesting that rFII has a proteolytic effect on human complements in vitro. 3.2. rFII prevents the deposition of complement-inudced membrane attack complexes of complement(MAC) on HUVECs We next evaluated the effects of rFII on deposition of MAC assembled by C5b-9 on HUVECs membrane. As Fig. 2 shown, an intensive MAC deposition was observed on the HUVECs membrane in normal group. Conversely, in rFII pre-treated groups, it was shown a decrease in MAC deposition on HUVECs membrane; however there was no change of the MAC deposition on HUVECs membrane in rFII post-treated group.
Fig. 1. Effect of rFII on human C5, C6, C9. Human complement C5, C6, C9 were incubated with/without rFII for 2 hours at 37 8C, the reaction was terminated by adding denature solution. The samples were analysised by SDS-PAGE (12%) and stained with coomassie brilliant blue. Lane 1: rFII; Lane 2: human fibrinogen; Lane 3: human fibrinogen + rFII; Lane 4: human complement C5; Lane 5: human complement C5 + rFII; Lane 6: human complement C9; Lane 7: human complement C9 + rFII; Lane 8: human complement C6; Lane 9: human complement C6 + rFII.
3.3. Degradation effect of rFII on complement in vivo To further determine if rFII can induce the degradation of complement in vivo, we detected the serum levels of C1q, C3 and C4 in rats after rFII infusion. As Table 2 shown, the serum levels of C1q, C3 and C4 in rats were progressively reduced in time- and dosedependent manners after rFII infusion, compared with the normal group (p < 0.05). 3.4. Protective effect of rFII against hyperacute rejection in an ex vivo perfusion model Given that the expand activation of complements plays a causal role in hyperacute rejection pathogenesis, we next evaluate whether rFII has a protective effect against HAR. In an ex vivo perfusion model, perfusion of human serum into the isolated rat hearts caused a significant decrease in the survival time (15.94 4.75 min) (Fig. 3 A), paralleled by a significant decrease in LVDP (Fig. 3C), the heart rate (Fig. 3B), and the level of C1q, C3, C4 (Table 3). In contrast, in the complement-inactive serum perfusion group, the heart rate and LVDP dropped slowly (Fig. 3B and C) and the survival time was 94.62 23.91 min (Fig. 3A) while the concentration of the complement before and after experiment had no difference statistically. Analogous to the complement-inactive serum perfusion, in rFII treatment group, the rate of heart and the LVDP also dropped slowly when administration of 10, 20, 50 mg/ml rFII before start, compared with human plasma perfusion group. However, the levels of complement C1q, C3 and C4 in perfusates were significant reduced. The survival time were 44.24 13.97 min, 74.21 20.88 min, and 83.36 16.63 min, respectively. These findings imply that rFII may have a anti-HAR role in vivo.
Table 1 rFII treated and untreated SD rat serums for different times (2 h, 6 h and 12 h). The quantity of complement required for 50% hemolysis is defined as CH50 value. The data shown below was converted to percentage with a value of 100% assuned for basal data. Group
2 h (%)
6 h (%)
12 h (%)
Normal rFII (10 ug/ml) rFII (20 ug/ml) rFII (50 ug/ml)
98.16 5.31 71.73 6.75* 63.19 7.41* 42.58 5.82*
100.63 6.12 48.71 5.18* 29.67 4.11* 15.74 4.01*
101.66 6.37 34.30 5.98* 18.12 4.09* 5.22 2.26*
*
p < 0.05 as compared to the normal group. Data are presented as the mean S.D.
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Fig. 2. The degradation effect of rFII on MAC in HUVECs. Immunofluorescence assay analyzing the effect of rFII on MAC on the membranes of HUVECs. Blue: hochest 33258 staining; Red: MAC. MAC deposition was decreased in the rFII pre-treated groups, meanwhile, there was no change of the MACdeposition in rFII post-treated group.
3.5. Protective effect of rFII against HAR in the guinea pig-to-rat heart xenotransplant To demonstrate the anti-HAR effect of rFII in vivo, we use the guinea pig-to-rat heart xenotransplant model for further study. As Fig. 4A shown, the xenotransplant hearts treated with saline
caused the hearts in death rapidly. In addition, rejected organs showed areas of myocardial infarction associated with vascular thrombosis as well as the widespread deposition within vessels of immunoglobulins complement C3 (Fig. 4B and C). Conversely, the infusion of rFII before heart xenotransplant could significantly prolonged the survival time of heart in a dose-dependent manner.
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Table 2 Effects of rFII on the suppressions of C1q, C3, C4 in rats The plasma of rats in nomal and rFII- treated groups was collected for different times. Levels of C1q, C3, C4 were shown. The data shown below was converted to percentage with a value of 100% assuned for basal data. Groups
Time (h)
C1q (%)
C3 (%)
C4 (%)
Normal
2 4 6 2 4 6 2 4 6 2 4 6
102.65 8.36 95.31 11.27 94.90 12.31 89.18 15.05 63.64 17.38* 57.31 20.65* 80.33 21.68* 60.12 18.01* 35.21 14.57* 62.63 21.69* 38.12 16.87* 25.64 12.09*
101.10 8.31 101.17 10.61 97.89 10.45 91.97 20.31 72.87 22.98* 54.31 18.54* 73.67 25.75* 54.10 20.92* 28.37 13.16* 71.67 23.61* 40.78 19.01* 23.04 11.21*
99.97 9.75 104.32 8.06 102.18 13.68 98.58 12.73 72.31 15.90* 58.41 13.09* 79.14 20.01* 48.58 19.32* 26.49 13.73* 68.14 22.95* 36.66 16.99* 22.83 10.54*
0.1 mg/kg rFII
0.2 mg/kg rFII
0.5 mg/kg rFII
*
p < 0.05 as compared to the normal group. Data are presented as the mean S.D.
Consistently, the myocardial infarction associated with vascular thrombosis and the complement C3 deposition was also reduced. Furthermore, the serum levels of complement C1q, C3, C4 were significant decreased as well (Table 4). 4. Discussion In the present study, we reported that rFII from Agkistrodon acutus venom fraction could strongly induce the degradation of complements and inhibit the deposition of MAC in vitro, thereby protect against HAR both in an ex vivo perfusion model and an in vivo heterotopic guinea-pig-heart transplantation model. Organs transplanted between phylogenetically disparate species are subject to hyperacute rejection (HAR) characterized by intragraft deposition of preformed recipient immunoglobulin M (IgM) antibodies with subsequent complement activation [15]. Accumulated evidences indicated that the binding of xenoreative natural antibodies (XNAs) to the xenograft and the uncontrolled activation of complement system constitute the hallmark of HAR in xenotransplantation, which resulted in platelet aggregation,
Table 3 Effects of rFII on the complement C1q, C3, C4 in human plasma in the ex-vivo perfusion rat heart model. Five groups were established: human plasma group, complementinactive human plasma group, rFII(10 mg/ml, 20 mg/ml, 50 mg/ml, respectively) groups. Levels of C1q, C3, C4 were shown. Groups
Time
C1q(mg/L)
C3(mg/L)
C4(mg/L)
Human plasma
Pre-infusion Post-infusion Pre-infusion Post-infusion Pre-infusion Post-infusion Pre-infusion Post-infusion Pre-infusion Post-infusion
78.67 28.56 36.73 18.67* 15.21 8.67* 11.72 5.36* 53.21 20.30* 30.98 13.74* 38.53 18.69* 25.74 11.31* 20.36 7.96* 17.64 6.32*
542.74 74.32 158.96 44.73* 36.77 18.90* 34.75 16.73* 213.93 56.74* 108.32 37.89* 115.32 37.08* 83.08 27.16* 75.73 21.74* 69.74 17.68*
212.77 63.81 94.66 37.21* 25.32 10.87* 25.64 10.74* 86.33 40.10* 50.78 23.19* 53.21 24.67* 39.36 11.78* 43.77 16.09* 38.18 12.97*
Complement-inactive human plasma 10 mg/ml rFII 20 mg/ml rFII 50 mg/ml rFII *
P < 0.05 as compared to human-plasma group. Datas are presented as the mean S.D.
Fig. 3. Effects of rFII on survival rates, heart rate and LVDP in ex-vivo rat hearts perfused with human plasma. (A) Survival time of isolated rat hearts was tested immediately after ex-vivo perfusion of human serum.*P < 0.05 as compared to the human plasma group. (B) Effect of rFII on the heart rat in the ex-vivo perfusion model. (C) Effect of rFII on the LVDP. Values of heart rate and LVDP are expressed as the mean S.D. *P < 0.05, **P < 0.01 as compared to human serum group.
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Fig. 4. Effects of rFII against HAR in the guinea pig-to-rat heart xenotransplant. (A) Four groups were established, rFII-treated groups(low-, medium-, and high-dose) were given different doses of rFII(10 mg/ml, 20 mg/ml, 50 mg/ml), normal group was infused with saline. Survival time of donor hearts was tested after transplantation. *P < 0.05 as compared to the HAR group. (B) Effect of rFII on myocardial infarction in the guinea pig-to-rat heart xenotransplant. The infusion of rFII before heart xenotransplant reduced the myocardial infarction associated with vascular thrombosis in dose-dependent manner. (C) Effect of rFII on complement C3 deposition in the guinea pig-to-rat heart xenotransplant. The infusion of rFII before heart xenotransplant reduced the C3 deposition in dose-dependent manner.
coagulation and in turn disruption of vascular endothelial integrity with loss of endothelial functions, finally leading to irreversible xenograft injury and destruction within minutes to a few hours [16,17]. Prevention of this rapid reaction has been considered to be the first step for successful xenotransplantaiton. It is reported that
both prostacyclin and glycoprotein IIb/IIa can protect against HAR in a rabbit-to dog lung xenotransplatant model [18]. Furthermore, the combination therapy of SCR1(soluble human complement receptor type 1) and ATIII(antithrombin III) can also prolong xenograft survival in HAR [19]. Therefore, inhibition of both
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Table 4 Effects of rFII on the complement C1q, C3, C4 in serum the guinea pig-to-rat heart xenotransplant. Four groups were established: HAR group, rFII(0.1 mg/kg, 0.2 mg/kg, 0.5 mg/ kg, respectively) groups. Levels of C1q, C3, C4 were shown. Groups
Time
C1q(mg/L)
C3(mg/L)
C4(mg/L)
HAR
Pre-explanted Post-explanted Pre-explanted Post-explanted Pre-explanted Post-explanted Pre-explanted Post-explanted
0.154 0.076 0.063 0.026* 0.073 0.098* 0.046 0.012* 0.041 0.014* 0.036 0.012* 0.037 0.011* 0.024 0.012*
0.411 0.108 0.189 0.077* 0.219 0.075* 0.147 0.032* 0.173 0.065* 0.096 0.040* 0.073 0.024* 0.068 0.017*
0.671 0.137 0.273 0.098* 0.357 0.112* 0.243 0.106* 0.278 0.089* 0.196 0.071* 0.187 0.068* 0.175 0.072*
0.1 mg/kg rFII 0.2 mg/kg rFII 0.5 mg/kg rFII *
P < 0.05 as compared to the HAR control group. Datas are presented as mean S.D.
complement activation and coagulation cascade seems to be a reasonable way to avoid HAR. In our previous study [10,11,20], we characterized the hydrolytic activity of rFII, and found that rFII could degrade the fibrin/fibrinogen in vitro, dissolve the thrombi/ microthrombi in vivo, and inhibit platelet aggregation triggered by ADP(adenosine diphosphate) in human platelet rich plasma. Based on these properties, rFII could significantly decrease the TAT in vitro and dissolve the thrombi in xenografts. It is known that the onset of complement activation can forecast xenograft rejection in HAR [1]. Histological findings have demonstrated that there is a large number of C3 and C5b-9 deposition on the cell membrane in xenografts in HAR [1]. In our present study, the effect of rFII on HAR was investigated in the guinea pig-to-rat heart xenotransplantation model as well as the ex vivo perfusion model. We found that rFII could significantly prolong the isolated heart survival time and improve the cardiac function. The dramatic anti-HAR effect of rFII may rely on two possible mechanisms. Firstly, the protective effect of rFII was mainly due to the strong anti-complement activity. rFII could decrease the CH50 activity in vitro and the levels of C1q, C3, and C4 in vivo; In addition, SDS-PAGE analysis also showed that the human complements(C5, C6, and C9) were largely degraded after preincubation with 10 ug/ml rFII, indicating that rFII could inhibit the complement activity via degradation of complements and meliorate the damage of xenografts induced by complement activation; Furthermore, the anti-MAC deposition effect of rFII may be the consequence of its anti-complement activity. It is known that the development of HAR depends on assembly of the terminal C5-C9 complement pathway [21]. In this investigation, the deposition of MAC on HUVECs membrane was almost disappeared in rFII preincubated group, compared with normal group, however no change of the intension of MAC on HUVECs was observed in rFII posttreatmented group. These findings suggested that rFII could inhibit the assembling of MAC, but have no influence on the assembled MAC. Secondly, we have demonstrated that rFII can directly lyses fibrin without activating intrinsic plasminogen and effectively dissolved microthrombi [10,11,22]. It is increasingly proven that the intravascular thrombosis plays an important role in loss of xenograft [9] and microvacular thrombosis is one of the histological character features of HAR [1]. Thus, both the fibrinolytic activity and inhibition of platelet aggregation effect of rFII indicate that rFII might ameliorate the reduced blood supply to xegrafts and relieve the additional damage by thrombosis. Taken together, all these multiple roles of rFII are likely to cooperate with each other and contribute to the favorable therapeutic effect on HAR. Current therapeutic strategies for abrogation of HAR include pretransplant antibody absorption by specific or nonspecific extracorporeal column perfusion, ex vivo donor organ perfusion, the administration of substances interfering with complement activation, or even the genetic alteration of the donor [15]. Furthermore, some compounds or biological reagents have been
proven to be effective in overcoming HAR, such as soluble complement receptor 1,C1-inhibitor protein, soluble CD46,monoclonal antibodies to complement components C5 and C8, cobra venom factor(CVF), Hypodermin A, tirofiban and various combination of these compounds [18,23–29]. However, administration of these agents are also associated with the high risk of side-effects [8]. As for rFII, our previous investigation had convinced the safety of rFII was well with the LD50 value as higher as 53.5 mg/kg, compared with its concentration used in vivo as 0.5 mg/kg [11], suggesting that rFII might represent a novel agent of potential therapeutic effect for the prevention of hyperacute rejection. In conclusion, our present investigation reveals that rFII, a novel recombinant fibrinogenase from Agkistrodon acutus venom, can effectively protect against HAR through direct degradation of complements and inhibition of assembling of MAC. Acknowledgments Project was supported by the National Natural Science Fundation of China (No.81000209), the key project of Chinese Ministry of Education (No.210255), and the fundamental Research for the Central Universities (No.21609304). References [1] Yang YG, Sykes M. Xenotransplantation: current status and a perspective on the future. Nat Rev 2007;7:519–31. [2] Auchincloss Jr H. Xenogeneic transplantation. A review. Transplantation 1988;46:1–20. [3] Dalmasso AP, et al. Mechanism of complement activation in the hyperacute rejection of porcine organs transplanted into primate recipients. Am J Pathol 1992;140:1157–66. [4] Dalmasso AP. The complement system in xenotransplantation. Immunopharmacology 1992;24:149–60. [5] Baldwin 3rd WM, Pruitt SK, Brauer RB, Daha MR, Sanfilippo F. Complement in organ transplantation. Contributions to inflammation, injury, and rejection. Transplantation 1995;59:797–808. [6] Galbusera M, et al. Activation of porcine endothelium in response to xenogeneic serum causes thrombosis independently of platelet activation. Xenotransplantation 2005;12:110–20. [7] Buhler L, et al. Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates. Transplantation 2000;70:1323–31. [8] Cozzi E, et al. An update on xenotransplantation. Vet Res Commun 2007;31(Suppl. 1):15–25. [9] Cowan PJ, d’Apice AJ. Complement activation and coagulation in xenotransplantation. Immunol Cell Biol 2009;87:203–8. [10] Wang R, et al. Novel recombinant fibrinogenase of Agkistrodon acutus venom protects against LPS-induced DIC. Thromb Res 2009;123:919–24. [11] Wang R, et al. Recombinant fibrinogenase from Agkistrodon acutus venom protects against sepsis via direct degradation of fibrin and TNF-alpha. Biochem Pharmacol 2008;76:620–30. [12] Harrison RA, Lachman PJ. Complement technology. In: Wier DM, editor. Handbook of Experimental Immunology Immunochemistry, vol. 1. Oxford: Blackwell Scientific Publications; 1986. [13] Ono K, Lindsey ES. Improved technique of heart transplantation in rats. J Thoracic Cardiovasc Surgery 1969;57:225–9. [14] Deng J, et al. beta-catenin interacts with and inhibits NF-kappa B in human colon and breast cancer. Cancer Cell 2002;2:323–34.
X. Lin et al. / Biochemical Pharmacology 85 (2013) 772–779 [15] Loss M, et al. A primate model for discordant pig to primate kidney xenotransplantation without hyperacute graft rejection. J Invest Surg 2001;14:21–9. [16] Parker W, et al. Transplantation of discordant xenografts: a challenge revisited. Immunol Today 1996;17:373–8. [17] Platt JL, Bach FH. The barrier to xenotransplantation. Transplantation 1991;52:937–47. [18] Chen G, et al. Prevention of hyperacute rejection of pig-to-monkey cardiac xenografts by Chinese cobra venom factor. Transplant Proc 2001;33:3857–8. [19] Fujiwara I, Nakajima H, Akioka K, Matsuda T, Oka T. Soluble complement receptor type 1 and antithrombin-III combination therapy prolongs xenograft survival: the role of thrombin and prostacyclin in hyperacute rejection. Transplant Proc 1997;29:935–7. [20] Jiang W, Ma T, Su X, Qiu P, Yan G. Enzymatic activities and functional characterization of a novel recombinant snake venom proteinase from Agkistrodon acutus. Biochimie 2009;91:277–87. [21] Mollnes TE, Fiane AE. Xenotransplantation: how to overcome the complement obstacle? Mol Immunol 1999;36:269–76. [22] Wang YW, et al. Fibrin(ogen)olytic character of FIIa isolated from Agkistrodon acutus venom. Acta Pharmacol Sin 2005;26:691–5. [23] Brandl U, et al. Combining the hDAF transgene with the GP IIb/IIIa inhibitor tirofiban improves heart performance and reduces myocardial damage
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following hyperacute rejection in an ex vivo perfusion model. Transplant Proc 2005;37:491–2. Chung KY, Park JJ, Han KH. Pig to canine auxiliary hepatic xenotransplantation model: prevention of hyperacute rejection via Kupffer cell blockade and complement regulation. Transplant Proc 2008;40:2755–9. Fiane AE, et al. C1-inhibitor attenuates hyperacute rejection and inhibits complement, leukocyte and platelet activation in an ex vivo pig-to-human perfusion model. Immunopharmacology 1999;42:231–43. Kroshus TJ, et al. Complement inhibition with an anti-C5 monoclonal antibody prevents acute cardiac tissue injury in an ex vivo model of pig-to-human xenotransplantation. Transplantation 1995;60:1194–202. Malassagne B, et al. Hypodermin A. a new inhibitor of human complement for the prevention of xenogeneic hyperacute rejection. Xenotransplantation 2003;10:267–77. Pruitt S, et al. Continuous complement (C) inhibition using soluble C receptor type 1 (sCR1): effect on hyperacute rejection (HAR) of pig-to-primate cardiac xenografts. Transplant Proc 1996;28:756. Rollins SA, Matis LA, Springhorn JP, Setter E, Wolff DW. Monoclonal antibodies directed against human C5 and C8 block complement-mediated damage of xenogeneic cells and organs. Transplantation 1995;60:1284–92.
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A novel recombinant fibrinogenase of Agkistrodon acutus venom protects against hyperacute rejection via degradation of complements Xi Lin a,1,*, Jie-zhen Qi b,1, Ming-hui Chen c, Bi-tao Qiu d, Zhen-hua Huang b, Peng-xin Qiu b, Jia-shu Chen b,**, Guang-mei Yan b,*** a
Department of Pharmacology, Medical College, Ji-Nan University, 601 Huangpu Road, Guangzhou, Guangdong 510632, PR China Department of Pharmacology, Zhong-Shan Medical College, Sun Yat-sen University, 74 Zhongshan Road II, Guangzhou, Guangdong 510089, PR China Department of Gynecology and Obstetrics, First Affiliated Hospital of Sun Yai-sen University, 58 Zhongshan Road II, Guangzhou, Guangdong 510080, PR China d School of Biology Sun Yat-sen University, 135 Xingangxi Road, Guangzhou, Guangdong 510275, PR China b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 September 2012 Accepted 13 November 2012 Available online 23 November 2012
Hyperacute rejection (HAR) is a main barrier in xenotransplantation, which is mediated by the combination of natural antibody to the xenograft and complement activation. Current therapies have focus on the inhibition of complement by development of complement inhibitor and transgenic animal organ. Here, we investigated the effects of rFII, a recombinant fibrinogenase from Agkistrodon acutus venom, on complement and HAR. The degradation effect of rFII on complement was tested by SDS-PAGE, CH50 examination, ELISA Kit and cofocal immunofluorescence microscopy in vitro and in vivo. An exvivo rat-to-human perfusion model and a vivo guinea-pig-to-rat heat HAR model were used to determine the protection of rFII against HAR. Our investigation indicated that rFII could significantly degrade human C5, C6, and C9, decrease the activity of complement, and inhibit the MAC deposition on HUVECs membrane in vitro. In addition, serum levels of C1q, C3 and C4 in rat were gradually reduced after infusion of rFII. Importantly, in an ex vivo rat-to-human perfusion model, the survival of rat hearts perfused with human serum treated with rFII (83.36 16.63 min) were significantly longer than that of hearts perfused with fresh human serum(15.94 4.75 min). At the time of 15 minutes after perfusion, functions of hearts added with 50 ug/ml rFII sustained well with heart rates at 283 65.32 beats/minute and LVDP at 13.70 5.45 Kpa, while that of hearts perfused with fresh human serum were severely damaged by HAR with heart rates at 107.77 40.31 beats/minute and LVDP at 1.01 0.83 Kpa. We also found that rFII significantly decreased the levels of C1q, C3 and C4 in human fresh serum perfusate. In a vivo guinea-pig-torat heat HAR model, the survival of rat hearts treated with rFII were significantly longer than that of hearts perfused with normal saline; and relieved heart damage by complete activation. Our finding demonstrates the anti-complement property of rFII and its protection against HAR, indicating that rFII might be as a potential therapeutic agent for xenotransplantation. ß 2012 Elsevier Inc. All rights reserved.
Keywords: Xenotransplantation Hyperacute rejection Complement
1. Introduction
Abbreviations: ADP, adenosine diphosphate; ATII, antithrombin; CH50, complement hemolytic activity 50; CVF, cobra venom factor; HAR, hyperacute rejection; HUVEC, human umbilical vein endothelial cell; HR, heart rate; IgM, immunoglobulin M; LVDP, left ventricular developed pressure; LSCM, laser scanning confocal microscopy; MAC, membrane attack complex; rFII, recombinant fibrinogenase of Agkistrodon acutus venom; SCR1, soluble human complement receptor type1; XNAs, xenoreactive natural antibodies. * Corresponding author. Tel.: +8620 85220242; fax: +8620 85228865. ** Corresponding author. Tel.: +8620 87330553; fax: +8620 87330553. *** Corresponding author. Tel.: +8620 87330578; fax: +8620 87330578. E-mail addresses: [email protected] (X. Lin), [email protected] (J.-s. Chen), [email protected] (G.-m. Yan). 1 Co-first authors. 0006-2952/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2012.11.012
Clinical transplantation is the only effective therapy for endstage organ failure. Unfortunately, many patients do not receive this therapy owing to the severe shortage of suitable human organ donors [1]. This supply-demand imbalance could be corrected by transplanting organs from other species (xenografts) into human. The most major barrier to xenotransplantation, however, is hyperacute rejection (HAR) to the organ graft. HAR is mediated by the binding of preexisting of xenoreactive natural antibodies(XNAs) to antigens expressed on the donor organ endothelia, which active complement and finally lead to thrombosis and destruction to the graft [2,3]. The antibody and complement system have been shown to play a central pathophysiologic role in HAR and to contribute to the inflammation and
X. Lin et al. / Biochemical Pharmacology 85 (2013) 772–779
organ injury associated with transplantation [4,5]. Furthermore, increasing evidence suggested that coagulation and thrombotic disorders were associated with HAR as well, which was characterized by the formation of platelet adhesion, aggregation, thrombosis, and endothelial dysfunction resulting from activation of the coagulation pathway [6,7]. Thus, interventions made to overcome HAR mainly concentrated on prevention of complement activation or removal xenoreactive natural antibodies [1,8] and therapeutic strategies to prevent intravascular thrombosis after xenotransplantation have received increasing attention as well. Despite these advances, development of an effective drug that has therapeutic role against HAR is still in urgent. Our previous results revealed that rFII, a novel recombinant fibrinogenase from Agkistrodon acutus venom could inhibit the platelet aggregation and directly degrade the microvascular thrombosis without activating intrinsic plasminogen [9–11]. Based on the hypothesis that inhibition of both complememnts and coagulation cascade could be a clue to avoid HAR, an exploration for the possible therapeutic potential of rFII against HAR is therefore essential. Here we demonstrated that rFII cleavaged and decreased the activity of complements in vitro, reduced the serum level of complements in vivo and protected against HAR in a rat heart ex vivo perfusion model and a guinea pig-to-rat heart HAR model, indicating that it might be an potential drug candidate against HAR. 2. Materials and methods 2.1. Reagents, cell cultures and animals The recombinant fibrinogenase II(rFII) from Agkistrodon acutus venom was prepared as we previously described [11]. Human Umbilical Vein Endothelial cells (HUVECs) were obtained from the American Type Culture Collection (Manassas, USA). Cells maintained in DMEM (Invitrogen, USA) supplemented with 10% FBS and a humidified atmosphere of 5% CO2 at 37 8C. All animal experiments were conducted in accordance with the National Guide for the Care and Use of Laboratory Animals and were approved by Sun Yat-sen University Animal Care and Use Committee (Guangzhou, China). Adult male S-D rats (250–300 g) and adult male guinea-pigs (300–350 g) were supplied by the Experimental Animal Center of Zhongshan Medical College, Sun Yat-san University. 2.1.1. Degradation effect of rFII on human complement in vitro Human complement C5, C6 and C9 (Sigma Aldrich, USA) were incubated with 10 ug/ml rFII at 37 8C for 2 h. Then samples were analysed by 12% SDS-PAGE. 2.1.2. Complement hemolytic activity (CH50) assay The complement hemolytic activity (CH50) was detected as previously described [12]. The red cells of guinea-pig were diluted to the concentration of 2% by the buffer (NaCl17g, Na2HPO4 11 g, KH2PO4 0.27 g, 10% MgCl2 2 ml and distilled water 280 ml). Then the dilutions were incubated with isovolumic hemolysin at 37 8C for 0.5 h. The SD rat serums were treated with or without rFII for different times (2 h, 6 h and 12 h). We established four groups: low-dose (10 mg/ml rFII) group, medium-dose(20 mg/ml rFII) group, high-dose(50 mg/ml rFII) group and normal group. The appropriate series of treated or untreated rat serum were added into a series of reaction tubes containing the buffer and sensitized guinea-pig red cells. After incubation at 37 8C for 1 h, the tubes were centrifuged to remove unlysed cells, and the optical density of supernatant was measured at 540 nm. The quantity of complement required for 50% hemolysis is defined as CH50 value.
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Data were expressed as the percent change in CH50 at selected time point which was calculated using the following formula: (CH50 value at the selected time point/pre-injury baseline value 100%). 2.1.3. Degradation effect of rFII on serum complement in vivo Rats were infused with rFII for 6 h at the speed of 1 ml/h through the right femoral vein. Four different groups were established: normal group: saline was infused; rFII groups: 0.1 mg/kg, 0.2 mg/kg and 0.5 mg/kg. The plasma of rats were collected in tube at pre-infusion (0 h) and post-infusion (2 h, 4 h and 6 h) time points and stored at 20 8C until assayed. The concentrations of C1q, C3 and C4 in animal plasma was determined using ELISA Kit (R&D, USA). 2.1.4. Ex-vivo rat hearts perfused with human serum supplemented with rFII This working heart–perfusion model used for hyperacute rejection was prepared as previously described [13]. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbitone (100 mg/kg). Heparin (200IU/100 g) was injected via the inferior vena cava, and the heart was quickly removed to ice-cold Krebs-Henseleit buffer (K-H buffer) containing (mM): NaCl 118, KCl 4.7, CaCl2 1.75, MgSO4 1.2, glucose 11, EDTA 0.5, NaHCO3 25. Following aortic cannulation, hearts were perfused in the ex vivo Langendorff mode at a constant flow rate of 15 ml/min with oxygenated (95%O2 and 5% CO2) perfusate at 37 8C. Two electrodes were put on the left ventricle and the right atrium to monitor heart rate (HR) and left ventricular developed pressure (LVDP). Human blood was obtained from healthy volunteers after informed consent, separated into serum and cellular components by centrifugation. The hearts of 60 male SD rats (200–300 g) were explanted. Administration was started after 30-min equilibration period. Six groups were established: serum-control group was perfused with fresh human serum and served as a positive control; decomplementation group was perfused with the decomplemented human serum which was performed by heating of the serum at 56 8C for 30 minutes to inactivate the complement activity as negative controls. rFII-treated groups (low-, medium-, and highdose rFII) were given the human serum which incubated with different doses of rFII(10 ug/ml, 20 ug/ml and 50 ug/ml) for 6 h at 37 8C before perfusion. The additional isolated hearts, which were given neither treated- nor untreated- human serum, were infused with K-H buffer. The experiments were terminated when hearts failed to pump against the afterload column of 55 mm Hg and the survival time of perfused hearts was recorded. The serum were collected in tube before and after experiments and stored at 20 8C The concentrations of C1q, C3 and C4 in human serum were determined using ELISA Kit (R&D,USA). 2.1.5. Guinea-Pig-to-Rat heart transplantation in vivo S-D rat underwent a heterotopic guinea-pig-heart transplantation into the abdomen as previously described [13]. The transplant was performed through a lower midline abdominal incision, implanting the guinea-pig heart by anastomosis of the donor aorta to the recipient abdominal aorta and of donor pulmonary artery to the recipient inferior vena cava. Treatments were started 6 h before heterotopic heart transplantation through femoral vein. Four groups were established, rFII-treated groups (low-, medium-, and high-dose rFII) were given different doses of rFII(0.1 mg/kg, 0.2 mg/kg, and 0.5 mg/kg) for 6 h. Normal group: saline was infused. Guinea-pig heart xenografts were removed at the time of rejection or death for histological study with H/E staining, and immunohistochemically for immunoglobulin C3.
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2.1.6. Confocal immunofluorescence microscopy of C5b-9 neoantigen We evaluated the effects of rFII on MAC(membrane attack complex) through assembling C5b-9 on the membranes of HUVECs. Assembly of sublytic C5b-9 on the membranes of HUVECs was done with using purified complement complex, including C5b6 (1.0 mg/ml), C7 (10 mg/ml), C8 (10 mg/ml) and C9 (10 mg/ml) according to the manufacture’s instructions (Calbiochem, USA). Four groups were established: normal control group, pre-rFII groups (1, 10 mg/ml, respectively), post-rFII group (10 mg/kg). Laser scanning confocal microscopy assay (LSCM) was used to assess C5b-9 neoantigen localization on membranes of HUVECs. Cells were fixed in a 1:1 methanol: acetone solution for 30 min following by blocking in goat serum for 1 h. 1:200 monoclonal anti-C5b-9 antibody (Invitrogen, USA) were added to the cells and incubated at 4 8C overnight. FITC-conjugated goat anti-rat IgG (Cell Signaling Technology, USA) was added for 1 h at room temperature and 1:1000 Hoechst33258 (Molecular Probes, USA) was added for 15 minutes in the dark and mounted to glass slides until visualized by confocal immunofluorescence microscopy (Nikon, Japan). 2.1.7. Histopathological Analysis Heart samples were fixed in 10% neutral-buffered formalin, embedded in paraffin, and stained with hematoxylin-eosin. 2.1.8. Immunohistochemistry Immunohistochemistry staining of 4 mm sections of paraffinembedded samples were performed as described [14]. Sections were stained with 1:200 primary rabbit anti-rat antibody C3 (invitrogen, USA) overnight at 4 8C, bio-anti rabbit IgG for 1 hour and then incubated with avidin biotin-peroxidase complex diluted in NaCl/Pi. 2.2. Statistical analysis Differences between data groups were assessed for significance using Student’s t-test of unpaired data or one-way analysis of variance. Repeated measures analysis of variance was used for multivariate analysis. All experiments were repeated at least three times and the data were presented as the mean S.D.
3. Results 3.1. Proteolytic effect of rFII on human complements in vitro To investigate whether rFII has a proteolytic effect on human complements in vitro, we tested the protein level of human complements after pre-incubation with rFII. SDS-PAGE analysis showed that the human complements(C5, C6, and C9) were dramatically degraded after pre-incubation with 10 ug/ml rFII for 2 h at 37 8C (Fig. 1). Consistently, the activity of complement was also significantly decreased as the time of pre-incubation with rFII prolonged (Table 1), suggesting that rFII has a proteolytic effect on human complements in vitro. 3.2. rFII prevents the deposition of complement-inudced membrane attack complexes of complement(MAC) on HUVECs We next evaluated the effects of rFII on deposition of MAC assembled by C5b-9 on HUVECs membrane. As Fig. 2 shown, an intensive MAC deposition was observed on the HUVECs membrane in normal group. Conversely, in rFII pre-treated groups, it was shown a decrease in MAC deposition on HUVECs membrane; however there was no change of the MAC deposition on HUVECs membrane in rFII post-treated group.
Fig. 1. Effect of rFII on human C5, C6, C9. Human complement C5, C6, C9 were incubated with/without rFII for 2 hours at 37 8C, the reaction was terminated by adding denature solution. The samples were analysised by SDS-PAGE (12%) and stained with coomassie brilliant blue. Lane 1: rFII; Lane 2: human fibrinogen; Lane 3: human fibrinogen + rFII; Lane 4: human complement C5; Lane 5: human complement C5 + rFII; Lane 6: human complement C9; Lane 7: human complement C9 + rFII; Lane 8: human complement C6; Lane 9: human complement C6 + rFII.
3.3. Degradation effect of rFII on complement in vivo To further determine if rFII can induce the degradation of complement in vivo, we detected the serum levels of C1q, C3 and C4 in rats after rFII infusion. As Table 2 shown, the serum levels of C1q, C3 and C4 in rats were progressively reduced in time- and dosedependent manners after rFII infusion, compared with the normal group (p < 0.05). 3.4. Protective effect of rFII against hyperacute rejection in an ex vivo perfusion model Given that the expand activation of complements plays a causal role in hyperacute rejection pathogenesis, we next evaluate whether rFII has a protective effect against HAR. In an ex vivo perfusion model, perfusion of human serum into the isolated rat hearts caused a significant decrease in the survival time (15.94 4.75 min) (Fig. 3 A), paralleled by a significant decrease in LVDP (Fig. 3C), the heart rate (Fig. 3B), and the level of C1q, C3, C4 (Table 3). In contrast, in the complement-inactive serum perfusion group, the heart rate and LVDP dropped slowly (Fig. 3B and C) and the survival time was 94.62 23.91 min (Fig. 3A) while the concentration of the complement before and after experiment had no difference statistically. Analogous to the complement-inactive serum perfusion, in rFII treatment group, the rate of heart and the LVDP also dropped slowly when administration of 10, 20, 50 mg/ml rFII before start, compared with human plasma perfusion group. However, the levels of complement C1q, C3 and C4 in perfusates were significant reduced. The survival time were 44.24 13.97 min, 74.21 20.88 min, and 83.36 16.63 min, respectively. These findings imply that rFII may have a anti-HAR role in vivo.
Table 1 rFII treated and untreated SD rat serums for different times (2 h, 6 h and 12 h). The quantity of complement required for 50% hemolysis is defined as CH50 value. The data shown below was converted to percentage with a value of 100% assuned for basal data. Group
2 h (%)
6 h (%)
12 h (%)
Normal rFII (10 ug/ml) rFII (20 ug/ml) rFII (50 ug/ml)
98.16 5.31 71.73 6.75* 63.19 7.41* 42.58 5.82*
100.63 6.12 48.71 5.18* 29.67 4.11* 15.74 4.01*
101.66 6.37 34.30 5.98* 18.12 4.09* 5.22 2.26*
*
p < 0.05 as compared to the normal group. Data are presented as the mean S.D.
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Fig. 2. The degradation effect of rFII on MAC in HUVECs. Immunofluorescence assay analyzing the effect of rFII on MAC on the membranes of HUVECs. Blue: hochest 33258 staining; Red: MAC. MAC deposition was decreased in the rFII pre-treated groups, meanwhile, there was no change of the MACdeposition in rFII post-treated group.
3.5. Protective effect of rFII against HAR in the guinea pig-to-rat heart xenotransplant To demonstrate the anti-HAR effect of rFII in vivo, we use the guinea pig-to-rat heart xenotransplant model for further study. As Fig. 4A shown, the xenotransplant hearts treated with saline
caused the hearts in death rapidly. In addition, rejected organs showed areas of myocardial infarction associated with vascular thrombosis as well as the widespread deposition within vessels of immunoglobulins complement C3 (Fig. 4B and C). Conversely, the infusion of rFII before heart xenotransplant could significantly prolonged the survival time of heart in a dose-dependent manner.
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Table 2 Effects of rFII on the suppressions of C1q, C3, C4 in rats The plasma of rats in nomal and rFII- treated groups was collected for different times. Levels of C1q, C3, C4 were shown. The data shown below was converted to percentage with a value of 100% assuned for basal data. Groups
Time (h)
C1q (%)
C3 (%)
C4 (%)
Normal
2 4 6 2 4 6 2 4 6 2 4 6
102.65 8.36 95.31 11.27 94.90 12.31 89.18 15.05 63.64 17.38* 57.31 20.65* 80.33 21.68* 60.12 18.01* 35.21 14.57* 62.63 21.69* 38.12 16.87* 25.64 12.09*
101.10 8.31 101.17 10.61 97.89 10.45 91.97 20.31 72.87 22.98* 54.31 18.54* 73.67 25.75* 54.10 20.92* 28.37 13.16* 71.67 23.61* 40.78 19.01* 23.04 11.21*
99.97 9.75 104.32 8.06 102.18 13.68 98.58 12.73 72.31 15.90* 58.41 13.09* 79.14 20.01* 48.58 19.32* 26.49 13.73* 68.14 22.95* 36.66 16.99* 22.83 10.54*
0.1 mg/kg rFII
0.2 mg/kg rFII
0.5 mg/kg rFII
*
p < 0.05 as compared to the normal group. Data are presented as the mean S.D.
Consistently, the myocardial infarction associated with vascular thrombosis and the complement C3 deposition was also reduced. Furthermore, the serum levels of complement C1q, C3, C4 were significant decreased as well (Table 4). 4. Discussion In the present study, we reported that rFII from Agkistrodon acutus venom fraction could strongly induce the degradation of complements and inhibit the deposition of MAC in vitro, thereby protect against HAR both in an ex vivo perfusion model and an in vivo heterotopic guinea-pig-heart transplantation model. Organs transplanted between phylogenetically disparate species are subject to hyperacute rejection (HAR) characterized by intragraft deposition of preformed recipient immunoglobulin M (IgM) antibodies with subsequent complement activation [15]. Accumulated evidences indicated that the binding of xenoreative natural antibodies (XNAs) to the xenograft and the uncontrolled activation of complement system constitute the hallmark of HAR in xenotransplantation, which resulted in platelet aggregation,
Table 3 Effects of rFII on the complement C1q, C3, C4 in human plasma in the ex-vivo perfusion rat heart model. Five groups were established: human plasma group, complementinactive human plasma group, rFII(10 mg/ml, 20 mg/ml, 50 mg/ml, respectively) groups. Levels of C1q, C3, C4 were shown. Groups
Time
C1q(mg/L)
C3(mg/L)
C4(mg/L)
Human plasma
Pre-infusion Post-infusion Pre-infusion Post-infusion Pre-infusion Post-infusion Pre-infusion Post-infusion Pre-infusion Post-infusion
78.67 28.56 36.73 18.67* 15.21 8.67* 11.72 5.36* 53.21 20.30* 30.98 13.74* 38.53 18.69* 25.74 11.31* 20.36 7.96* 17.64 6.32*
542.74 74.32 158.96 44.73* 36.77 18.90* 34.75 16.73* 213.93 56.74* 108.32 37.89* 115.32 37.08* 83.08 27.16* 75.73 21.74* 69.74 17.68*
212.77 63.81 94.66 37.21* 25.32 10.87* 25.64 10.74* 86.33 40.10* 50.78 23.19* 53.21 24.67* 39.36 11.78* 43.77 16.09* 38.18 12.97*
Complement-inactive human plasma 10 mg/ml rFII 20 mg/ml rFII 50 mg/ml rFII *
P < 0.05 as compared to human-plasma group. Datas are presented as the mean S.D.
Fig. 3. Effects of rFII on survival rates, heart rate and LVDP in ex-vivo rat hearts perfused with human plasma. (A) Survival time of isolated rat hearts was tested immediately after ex-vivo perfusion of human serum.*P < 0.05 as compared to the human plasma group. (B) Effect of rFII on the heart rat in the ex-vivo perfusion model. (C) Effect of rFII on the LVDP. Values of heart rate and LVDP are expressed as the mean S.D. *P < 0.05, **P < 0.01 as compared to human serum group.
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Fig. 4. Effects of rFII against HAR in the guinea pig-to-rat heart xenotransplant. (A) Four groups were established, rFII-treated groups(low-, medium-, and high-dose) were given different doses of rFII(10 mg/ml, 20 mg/ml, 50 mg/ml), normal group was infused with saline. Survival time of donor hearts was tested after transplantation. *P < 0.05 as compared to the HAR group. (B) Effect of rFII on myocardial infarction in the guinea pig-to-rat heart xenotransplant. The infusion of rFII before heart xenotransplant reduced the myocardial infarction associated with vascular thrombosis in dose-dependent manner. (C) Effect of rFII on complement C3 deposition in the guinea pig-to-rat heart xenotransplant. The infusion of rFII before heart xenotransplant reduced the C3 deposition in dose-dependent manner.
coagulation and in turn disruption of vascular endothelial integrity with loss of endothelial functions, finally leading to irreversible xenograft injury and destruction within minutes to a few hours [16,17]. Prevention of this rapid reaction has been considered to be the first step for successful xenotransplantaiton. It is reported that
both prostacyclin and glycoprotein IIb/IIa can protect against HAR in a rabbit-to dog lung xenotransplatant model [18]. Furthermore, the combination therapy of SCR1(soluble human complement receptor type 1) and ATIII(antithrombin III) can also prolong xenograft survival in HAR [19]. Therefore, inhibition of both
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Table 4 Effects of rFII on the complement C1q, C3, C4 in serum the guinea pig-to-rat heart xenotransplant. Four groups were established: HAR group, rFII(0.1 mg/kg, 0.2 mg/kg, 0.5 mg/ kg, respectively) groups. Levels of C1q, C3, C4 were shown. Groups
Time
C1q(mg/L)
C3(mg/L)
C4(mg/L)
HAR
Pre-explanted Post-explanted Pre-explanted Post-explanted Pre-explanted Post-explanted Pre-explanted Post-explanted
0.154 0.076 0.063 0.026* 0.073 0.098* 0.046 0.012* 0.041 0.014* 0.036 0.012* 0.037 0.011* 0.024 0.012*
0.411 0.108 0.189 0.077* 0.219 0.075* 0.147 0.032* 0.173 0.065* 0.096 0.040* 0.073 0.024* 0.068 0.017*
0.671 0.137 0.273 0.098* 0.357 0.112* 0.243 0.106* 0.278 0.089* 0.196 0.071* 0.187 0.068* 0.175 0.072*
0.1 mg/kg rFII 0.2 mg/kg rFII 0.5 mg/kg rFII *
P < 0.05 as compared to the HAR control group. Datas are presented as mean S.D.
complement activation and coagulation cascade seems to be a reasonable way to avoid HAR. In our previous study [10,11,20], we characterized the hydrolytic activity of rFII, and found that rFII could degrade the fibrin/fibrinogen in vitro, dissolve the thrombi/ microthrombi in vivo, and inhibit platelet aggregation triggered by ADP(adenosine diphosphate) in human platelet rich plasma. Based on these properties, rFII could significantly decrease the TAT in vitro and dissolve the thrombi in xenografts. It is known that the onset of complement activation can forecast xenograft rejection in HAR [1]. Histological findings have demonstrated that there is a large number of C3 and C5b-9 deposition on the cell membrane in xenografts in HAR [1]. In our present study, the effect of rFII on HAR was investigated in the guinea pig-to-rat heart xenotransplantation model as well as the ex vivo perfusion model. We found that rFII could significantly prolong the isolated heart survival time and improve the cardiac function. The dramatic anti-HAR effect of rFII may rely on two possible mechanisms. Firstly, the protective effect of rFII was mainly due to the strong anti-complement activity. rFII could decrease the CH50 activity in vitro and the levels of C1q, C3, and C4 in vivo; In addition, SDS-PAGE analysis also showed that the human complements(C5, C6, and C9) were largely degraded after preincubation with 10 ug/ml rFII, indicating that rFII could inhibit the complement activity via degradation of complements and meliorate the damage of xenografts induced by complement activation; Furthermore, the anti-MAC deposition effect of rFII may be the consequence of its anti-complement activity. It is known that the development of HAR depends on assembly of the terminal C5-C9 complement pathway [21]. In this investigation, the deposition of MAC on HUVECs membrane was almost disappeared in rFII preincubated group, compared with normal group, however no change of the intension of MAC on HUVECs was observed in rFII posttreatmented group. These findings suggested that rFII could inhibit the assembling of MAC, but have no influence on the assembled MAC. Secondly, we have demonstrated that rFII can directly lyses fibrin without activating intrinsic plasminogen and effectively dissolved microthrombi [10,11,22]. It is increasingly proven that the intravascular thrombosis plays an important role in loss of xenograft [9] and microvacular thrombosis is one of the histological character features of HAR [1]. Thus, both the fibrinolytic activity and inhibition of platelet aggregation effect of rFII indicate that rFII might ameliorate the reduced blood supply to xegrafts and relieve the additional damage by thrombosis. Taken together, all these multiple roles of rFII are likely to cooperate with each other and contribute to the favorable therapeutic effect on HAR. Current therapeutic strategies for abrogation of HAR include pretransplant antibody absorption by specific or nonspecific extracorporeal column perfusion, ex vivo donor organ perfusion, the administration of substances interfering with complement activation, or even the genetic alteration of the donor [15]. Furthermore, some compounds or biological reagents have been
proven to be effective in overcoming HAR, such as soluble complement receptor 1,C1-inhibitor protein, soluble CD46,monoclonal antibodies to complement components C5 and C8, cobra venom factor(CVF), Hypodermin A, tirofiban and various combination of these compounds [18,23–29]. However, administration of these agents are also associated with the high risk of side-effects [8]. As for rFII, our previous investigation had convinced the safety of rFII was well with the LD50 value as higher as 53.5 mg/kg, compared with its concentration used in vivo as 0.5 mg/kg [11], suggesting that rFII might represent a novel agent of potential therapeutic effect for the prevention of hyperacute rejection. In conclusion, our present investigation reveals that rFII, a novel recombinant fibrinogenase from Agkistrodon acutus venom, can effectively protect against HAR through direct degradation of complements and inhibition of assembling of MAC. Acknowledgments Project was supported by the National Natural Science Fundation of China (No.81000209), the key project of Chinese Ministry of Education (No.210255), and the fundamental Research for the Central Universities (No.21609304). References [1] Yang YG, Sykes M. Xenotransplantation: current status and a perspective on the future. Nat Rev 2007;7:519–31. [2] Auchincloss Jr H. Xenogeneic transplantation. A review. Transplantation 1988;46:1–20. [3] Dalmasso AP, et al. Mechanism of complement activation in the hyperacute rejection of porcine organs transplanted into primate recipients. Am J Pathol 1992;140:1157–66. [4] Dalmasso AP. The complement system in xenotransplantation. Immunopharmacology 1992;24:149–60. [5] Baldwin 3rd WM, Pruitt SK, Brauer RB, Daha MR, Sanfilippo F. Complement in organ transplantation. Contributions to inflammation, injury, and rejection. Transplantation 1995;59:797–808. [6] Galbusera M, et al. Activation of porcine endothelium in response to xenogeneic serum causes thrombosis independently of platelet activation. Xenotransplantation 2005;12:110–20. [7] Buhler L, et al. Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates. Transplantation 2000;70:1323–31. [8] Cozzi E, et al. An update on xenotransplantation. Vet Res Commun 2007;31(Suppl. 1):15–25. [9] Cowan PJ, d’Apice AJ. Complement activation and coagulation in xenotransplantation. Immunol Cell Biol 2009;87:203–8. [10] Wang R, et al. Novel recombinant fibrinogenase of Agkistrodon acutus venom protects against LPS-induced DIC. Thromb Res 2009;123:919–24. [11] Wang R, et al. Recombinant fibrinogenase from Agkistrodon acutus venom protects against sepsis via direct degradation of fibrin and TNF-alpha. Biochem Pharmacol 2008;76:620–30. [12] Harrison RA, Lachman PJ. Complement technology. In: Wier DM, editor. Handbook of Experimental Immunology Immunochemistry, vol. 1. Oxford: Blackwell Scientific Publications; 1986. [13] Ono K, Lindsey ES. Improved technique of heart transplantation in rats. J Thoracic Cardiovasc Surgery 1969;57:225–9. [14] Deng J, et al. beta-catenin interacts with and inhibits NF-kappa B in human colon and breast cancer. Cancer Cell 2002;2:323–34.
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