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. Author manuscript; available in PMC: 2011 Sep 1.
Published in final edited form as: J Thromb Haemost. 2010 Sep;8(9):2001–2010. doi: 10.1111/j.1538-7836.2010.03950.x

ATORVASTATIN OR TRANSGENIC EXPRESSION OF TFPI INHIBITS COAGULATION INITIATED BY ANTI-NONGAL IgG BINDING TO PORCINE AORTIC ENDOTHELIAL CELLS

CC Lin *,†, M Ezzelarab *, H Hara *, C Long *, CW Lin *, A Dorling ‡, DKC Cooper *
PMCID: PMC2965779  NIHMSID: NIHMS219390  PMID: 20553382

Abstract

Background

Intravascular thrombosis remains a hurdle to successful xenotransplantation. Tissue factor (TF) expression on porcine aortic endothelial cells (PAECs), which results from their activation by xenoreactive antibodies (Abs) to Galα1,3Gal (Gal) and subsequent complement activation, plays an important role.

Objectives

The present study aimed to clarify the role of Abs directed against nonGal antigens in the activation of PAECs to express functional TF and to investigate selected methods of inhibiting TF activity.

Methods

PAECs from wild-type (WT), α1,3-galactosyltransferase gene-knockout (GT-KO) pigs, or pigs transgenic for CD46 or tissue factor pathway inhibitor (TFPI) were incubated with naïve baboon serum (BS) or sensitized BS (with high anti-nonGal Ab levels). TF activity of PAECs was assessed.

Results

Only fresh, but not heat-inactivated (HI), naïve BS activated WT PAECs to express functional TF. Similarly, PAECs from CD46 pigs were resistant to activation by naïve BS, but not to activation by fresh or HI sensitized BS. HI sensitized BS also activated GT-KO PAECs to induce TF activity. TF expression on PAECs induced by anti-nonGal Abs was inhibited if serum was pretreated with (i) an anti-IgG Fab Ab or (ii) atorvastatin, or (iii) when PAECs were transgenic for TFPI.

Conclusions

Anti-nonGal IgG Abs activated PAECs to induce TF activity through a complement-independent pathway. This implies that GT-KO pigs expressing a complement-regulatory protein may be insufficient to prevent the activation of PAECs. Genetic modification with an ‘anticoagulant’ gene, e.g., TFPI, or a therapeutic approach, e.g., atorvastatin, will be required to prevent coagulation dysregulation after pig-to-primate organ transplantation.

Keywords: Atorvastatin, Coagulation, Tissue factor, Tissue factor pathway inhibitor, Xenotransplantation

INTRODUCTION

Xenotransplantation promises an unlimited supply of organs and cells for clinical use. Pigs are thought the most suitable source of xenografts [1]. However, the antibody-mediated immunologic barrier hinders the success of xenotransplantation. Acute humoral xenograft rejection (AHXR) associated with the development of a thrombotic microangiopathy has proven difficult to prevent or treat [2].

The expression and activity of tissue factor (TF) plays a significant role. In vivo expression of TF is upregulated in necrotic xenografts [3]. In vitro, TF on porcine aortic endothelial cells (PAECs) is activated by the binding of anti-pig antibodies (Abs) and complement activation [4]. Our previous study indicated that TF activity on PAECs was increased only in the presence of complement, but not by Abs alone [5]. Gollackner et al [4], however, described complement-independent induction of TF by elicited IgG reactive with nonGal epitopes.

The generation of pigs homozygous for α1,3-galactosyltransferase gene-knockout (GT-KO) has established the importance of the role of Abs directed to antigen targets other than Galα1,3Gal (Gal) - anti-nonGal Abs - in the initiation of coagulation in pig-to-primate xenotransplantation models. Even in the absence of the pathogenic effect of anti-Gal Abs, most pig grafts are still lost from thrombotic microangiopathy within weeks. It is likely that anti-nonGal Abs play a significant role [6].

In the present paper, we report our investigations aimed at preventing an increase in TF activity after activation of PAECs by baboon and human anti-nonGal Abs. TF pathway inhibitor (TFPI) is the crucial regulator of the coagulation pathway initiated by TF. In vitro, expression of TFPI on immortalized PAECs effectively prevented porcine TF-dependent clotting induced by TNFα [7]. Statins - inhibitors of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase - have been demonstrated to have immunomodulatory, anti-inflammatory, and anticoagulant actions in addition to reducing the synthesis of cholesterol [8]. We have clarified the role of anti-nonGal Abs in activating PAECs to express TF and induce TF activity, and have investigated the suppressive (anticoagulant) effect of (i) PAECs that express TFPI or (ii) treatment with atorvastatin.

MATERIALS AND METHODS

Pig cell sources

PAECs were collected from 4 sources, namely (i) wild-type (WT) or (ii) GT-KO pigs, or (iii) pigs transgenic for the complement-regulatory protein, CD46, or (iv) TFPI (all provided by Revivicor, Inc., Blacksburg, VA). They were all of blood type non-A (O). The WT, GT-KO, and TFPI transgenic pigs were of Large White/Landrace/Duroc cross-breed, but were not from identical clones; however, the only major difference was in the absence of Gal antigens in the GT-KO pigs [9] or the expression of TFPI. The CD46 transgenic pigs were derived from a different herd of Large White pigs [10]. Constructs of TFPI encoding three human Kunitz domains of TFPI-CD4-P-selectin, and in vitro characterization after expression on endothelial cells have been described previously [7, 11–12]. A human CD31 promoter was generated and cloned into the vector for microinjection [12]. The TFPI transgenic pigs were born alive and survived without hemorrhagic complications until euthanized at approximately 12 months of age [13]. Since PAECs lack Weibel-Palade bodies, human TFPI should have been constitutively expressed on the surface of endothelial cells. However, expression of TFPI was minimal in most cases. The pig with the best expression of TFPI on aortic endothelial cells was selected for the present study.

Cell culture

PAECs were obtained and cultured as previously described[5]. The phenotype of CD46 and TFPI transgenic PAECs was determined by flow cytometry using an anti-CD46-based FITC Ab (Serotec, Raleigh, NC) and an anti-human TFPI Ab (American Diagnostica, Stamford, CT), respectively. FITC-conjugated mouse IgG1 and unconjugated mouse IgG1 (Serotec) were used as isotype controls, and donkey anti-mouse FITC Ab (Sigma, Ronkonkoma, NY) was used as a secondary Ab. The phenotype of GT-KO PAECs was previously confirmed [14]. The PAECs harvested from CD46 transgenic pigs expressed a high level of CD46; those from TFPI transgenic pigs constitutively expressed a low level of TFPI on the surface (Figure 1).

Figure 1. Surface phenotype of PAECs from CD46 and TFPI transgenic pigs.

Figure 1

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PAECs were incubated with FTIC-conjugated anti-CD46 and anti-TFPI Abs, respectively, and analyzed by flow cytometry to determine their surface phenotypes (Shaded: isotype control; solid line: WT PAECs; dash line: transgenic PAECs; left: CD46; right: TFPI). Very high expression of CD46 was observed in CD46-transgenic pigs. More modest expression of TFPI was observed in TFPI-transgenic pigs.

Activation of PAECs

Adherent PAECs were pre-incubated with fresh or heat-inactivated (HI) baboon or human serum. PAECs were harvested by 0.5% trypsin (Gibco, Paisley, UK) for mRNA and recalcified clotting assay analysis. In some experiments, PAECs were pre-incubated with atorvastatin (Pfizer, New York, NY) with/without mevalonic acid (Sigma) for 16h before sera were added. Since atorvastatin inhibits the conversion of HMG-CoA to mevalonic acid, which is the rate-limiting step reaction in cholesterol synthesis, adding mevalonic acid to the culture system blocked the inhibitory effect of atorvastatin. In other experiments, baboon sera were pre-incubated with anti-human IgG Fab Ab (Abcam, Cambridge, MA) or dithiothreitol (Sigma) for 30min before incubation with PAECs.

Preparation of sera

Sera were collected from immunologically-naïve baboons or healthy human volunteers. Sensitized baboon serum was collected from a baboon that had previously received an aortic patch graft from a GT-KO pig without immunosuppressive therapy. The median total IgG levels of naïve and sensitized baboon serum were 1,151 and 1,315 mg/dL, respectively (P=0.07) (as measured in the central laboratory of the University of Pittsburgh Medical Center).

Baboon or human IgM and IgG antibody binding to PAECs

Binding of xenoreactive Abs to PAECs was determined by flow cytometry, as previously described [14]. Detection of IgM or IgG binding was performed by incubating the serum with FITC-conjugated goat anti-human IgM and IgG (Invitrogen) Abs for 30min in the dark at 4°C.

Recalcified clotting assay

PAECs (1 × 105) were suspended in 50μl Tris-buffered saline and mixed with 100μl of Factor VII (FVII)-deficient human plasma (Haematologic Technologies, Essen Junction, VT) in glass tubes (Corning, Corning, NY). One hundred microliters (100μl) of 25mm CaCl2 in Tris-buffered saline were added and the tube incubated at 37°C in a water bath; the time for a fibrin clot to form was measured, during which time the tubes were continuously agitated by tilting. In these assays, TF-dependent thrombin generation requires the participation of FVII; the clotting time is shortened when FVII is added. Therefore, the procedure was repeated with the addition of FVII (Haematologic Technologies) in a separate assay. The activity of TF was determined by a comparison (ratio) of the clotting times measured with/without FVII. In each assay, the clotting time was determined in triplicate, and the results were quantified from a standard curve prepared by a series of dilutions of soluble recombinant human TF (R&D, Minneapolis, MN) and expressed as procoagulant activity equivalent to ng human TF per 1 × 105 PAECs.

Quantitative reverse transcription polymerase chain reaction (RT-PCR)

Three micrograms (3μg) of total RNA from each sample was used for reverse transcription with an oligo dT and a Superscript III (Invitrogen) to generate first-strand cDNA. PCR mixture was prepared using SYBR Green PCR Master Mix (Applied Biosystems). Primers for porcine TF were 5’-TTTACCAACTCGCCCCCCTTC -3’ (forward) and 5’-AATGTGCCGTTCACCCTGACTAAG -3’ (reverse) Primers for porcine β-actin were 5’-CTCGATCATGAAGTGCGACTG -3’ (forward) and 5’-GTGATCTCCTTCTGCATCCTGTC-3’ (reverse) (Invitrogen). The sequences of the above primers were analyzed in triplicate. Thermal cycling conditions were 10min at 95°C to activate the Amplitaq Gold DNA polymerase, followed by 40 cycles for 15sec at 95°C, and for 1min at 60°C on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

Rho activity

Rho activation was determined by the pull-down assay. Cell lysates were added to Rhotekin-Rho binding domain beads (Cytoskeleton, Denver, CO) and incubated for 1h at 4°C. The agarose beads were electrophoresed in 12% SDS-PAGE. Western blotting was performed with rabbit anti-Rho A antibody (Santa Cruz Biotechnology, Santa Cruz, CA).

Akt phosphorylation

Western blot was carried out to determine Akt phosphorylation[15]. The protein (30 μg) from PAECs was electrophoresed in 10% SDS-PAGE gel. Membranes were incubated with the following primary antibodies -, rabbit anti-Akt (Santa Cruz Biotechnology) and anti-β-actin (Sigma), followed by anti-rabbit IgG HRP Abs (Sigma).

Statistical analysis

Data are presented as mean ± SEM or median (n<3) from 3 independent experiments. Significance of the difference between two groups was determined by paired Student’s t-test or by the Mann-Whitney U test (n<3). Values of P<0.05 were considered significant.

RESULTS

Naïve baboon serum activates PAECs to induce TF activity that is complement-dependent

PAECs were incubated with fresh or HI naïve baboon serum for 8h. WT PAEC were demonstrated to induce TF activity in the recalcified clotting assay only by fresh, but not HI, baboon serum (Figure 2A). This confirmed a previous study demonstrating that activation of PAECs is dependent on the presence of complement [4–5]. To determine whether PAECs expressing a complement-regulatory protein are resistant to activation by primate serum, CD46 PAECs were incubated with fresh naïve baboon serum; this failed to activate these PAECs to induce TF activity, indicating the effect of regulating complement activity (Figure 2A). The functional TF activity was compatible with the expression of TF mRNA measured by quantitative RT-PCR after PAECs were pre-incubated with serum for 4h (Figure 2B).

Figure 2. Naïve baboon serum activates PAECs to induce TF activity that is complement-dependent.

Figure 2

Figure 2

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A. WT (whites bars) or CD46 (gray bars) PAECs were incubated with medium (control) or (at a series of concentrations) fresh or HI naïve baboon serum (NBS). The TF activity on the surface of PAECs was determined by the recalcified clotting assay. Fresh, but not heat-inactivated (HI), naïve baboon serum activated WT PAECs to express TF activity. CD46 PAECs were resistant to activation by fresh NBS.

(Left panel: fresh naïve baboon serum [NBS]; right panel: HI baboon serum [HI NBS])

( #p<0.01 compared to control).

B. WT (white bars) or CD46 (grey bars) PAECs were incubated with medium (control) or 10% fresh NBS or HI NBS for 4h. TF expression of PAECs was determined by quantitative RT-PCR. (# p<0.01 compared to control). Fresh, but not HI, NBS resulted in an increase in TF expression on WT, but not CD46, PAECs.

Sensitized baboon serum activates PAECs to induce TF activity that is complement-independent

To determine the effect of anti-nonGal Abs on PAECs, serum was collected from 3 baboons that had become sensitized to aortic patch grafts from GT-KO pigs 4 weeks previously. Anti-nonGal Abs were measured by determining IgM and IgG binding to GT-KO PAECs by flow cytometry. Sensitized baboon serum demonstrated significantly higher titers of anti-nonGal Abs compared to naïve baboon serum (Figure 3A). The experiments were then carried out by incubating PAECs with fresh or HI sensitized baboon serum (Figure 3B). WT PAECs demonstrated TF activity in the recalcified clotting assay not only after exposure to fresh sensitized baboon serum, but also to HI sensitized baboon serum. Moreover, CD46 PAECs were not fully resistant to activation by fresh or HI sensitized baboon serum, though they did demonstrate some resistance (Figure 3B). This indicated that sensitized baboon serum with a high titer of anti-nonGal Abs was able to activate PAECs independent of the involvement of complement. This effect was mainly from the presence of anti-nonGal Abs since HI sensitized but not naive baboon serum activated GT-KO PAECs to express TF, though perhaps not to quite the same extent (Figure 3C). The TF activity demonstrated by the recalcified clotting assay was compatible with the RNA expression by quantitative RT-PCR (Figure 3D).

Figure 3. Sensitized baboon serum activates PAECs to induce TF activity that is complement-independent.

Figure 3

Figure 3

Figure 3

Figure 3

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A. IgM and IgG binding of heat-inactivated naïve baboon serum (HI NBS, n=15) or heat-inactivated sensitized baboon serum (HI SBS; n=3) to PAECs (WT [white bars] and GT-KO [grey bars]) were determined by flow cytometry. Binding to GT-KO PAECs represents binding of anti-nonGal Abs. There was much greater binding of IgG, but not IgM, when PAECs were incubated with HI SBS.

B. WT (white bars) or CD46 (grey bars) PAECs were incubated with medium (control) or fresh (left panel) or HI (right panel) sensitized baboon serum (SBS) at a series of concentrations for 8h. The TF activity on the surface of PAECs was determined by the recalcified clotting assay. Both fresh and HI SBS activated WT and CD46 PAECs to induce TF activity.

C. GT-KO PAECs were incubated with medium (control) and HI NBS (white bars) or HI SBS (grey bars). HI SBS, but not HI NBS, activated PAECs to express TF activity.

D. WT (white bars), CD46 (grey bars), or GT-KO (striped bars) PAECs were incubated with medium (control) or 10% fresh SBS or HI SBS for 4h. TF expression on PAECs was determined by quantitative RT-PCR. (# p<0.01 compared to control.) TF expression was increased on PAEC from all pigs, with greater expression after exposure to fresh SBS than to HI SBS.

Sensitized baboon serum anti-Gal Abs activate PAECs to induce TF activity through IgG binding

To determine whether it is anti-nonGal IgM or IgG Abs that activate PAECs to induce TF activity, dithiothreitol was used to eliminate IgM. IgG binding of sensitized baboon serum to PAECs remained unchanged, but IgM binding disappeared after HI sensitized baboon serum was pretreated with dithiothreitol for 30min (Figure 4A). HI sensitized baboon serum pretreated with dithiothreitol activated WT PAEC to demonstrate the same level of TF activity compared to non-treated serum (Figure 4B). This implied that the ability of anti-nonGal Abs to activate PAECs was unrelated to IgM, and presumably related to IgG. To investigate further, HI sensitized baboon serum was pretreated with an anti-human IgG Fab Ab for 30min at 4°C before incubation with PAECs. The anti-human IgG Fab Ab blocked the effect of HI sensitized baboon serum to induce WT PAEC TF activity (Figure 4C). Together, these results indicated that HI sensitized baboon serum activated PAECs to express TF through IgG, but not IgM.

Figure 4. Sensitized baboon serum anti-nonGal Abs activate PAECs to induce TF activity through IgG binding.

Figure 4

Figure 4

Figure 4

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A. Heat-inactivated sensitized baboon serum (HI SBS) was pre-incubated with dithiothreitol (DTT, 200μl/ml) to eliminate IgM. IgM or IgG binding to PAECs was determined by flow cytometry. (Shaded: isotype control; solid line: non-treated serum; broken line: serum treated with DTT).

B. WT PAECs were incubated with medium (control) or HI SBS, which has been pretreated with DTT for 30min. TF activity on the surface of PAECs was activated by HI SBS was unrelated to the pretreatment of DTT ( the level of IgM) by recalcified clotting assay.

C. The experiment performed in B was repeated with HI SBS that had been treated with an anti-IgG Fab Ab at various concentrations (5–20μg/ml) for 30min at 4°C. Anti-IgG Fab Ab blocked the effect of HI SBS (with high levels of anti-nonGal Abs), inactivating WT PAECS (* p<0.05 compared to control).

Human sera with high levels of natural anti-nonGal Abs activate PAECs to induce TF activity that is complement-independent

To determine the role of anti-nonGal Abs, human serum was collected from 15 volunteers. Anti-nonGal IgG Abs were determined by flow cytometry using GT-KO PAECs as target cells. HI human sera were used to activate WT PAECs to induce TF activity. Thirteen percent (13%, i.e., 2/15) of sera demonstrated very high titers of anti-nonGal Abs with an approximate 5–7-fold increase over the other sera (Figure 5A). These two HI human sera induced a high level of TF activity on WT PAECs (Figure 5A, B) and CD46 PAECs (Figure 5B) and blocked by IgG Fab Ab (Figure 5C).

Figure 5. Human sera with high levels of natural anti-nonGal Abs activate PAECs to induce TF activity that is complement-independent.

Figure 5

Figure 5

Figure 5

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A. The relationship between the titer of anti-nonGal IgG Abs in 20% heat-inactivated human sera (n=15) and induced TF activity on PAECs. IgG binding to GT-KO PAECs is indicated. The same HI HS were used to activate WT PAECs. TF activity on PAECs was determined by the recalcified clotting assay.

B. WT and CD46 PAECs were incubated with HI HS with high-titer anti-nonGal Abs (n=2) at various concentrations (2.5–10%). TF activity on WT and CD46 PAECs was induced after incubation with these two HS by the recalcified clotting assay. (#p<0.01 compared to control).

C. The experiment performed in Figure 4C was repeated with 10% HI HS (with high-titer anti-nonGal Abs) that had been treated with an anti-Fab Ab at 20μg/ml. Anti-IgG Fab Ab blocked the effect of this human serum to activate WT PAECs to express TF.

Activation of PAECs by naïve and sensitized baboon sera to induce TF activity is inhibited by expression of TFPI

The above experiments were repeated using PAECs transgenic for TFPI. TF activity induced by fresh naïve baboon serum and by fresh or HI sensitized baboon serum was significantly attenuated by TFPI expression on PAECs (Figure 6A). The inhibitory effect of TFPI PAECs disappeared when an anti-TFPI Ab was present (Figure 6A). However, after incubation with baboon serum, mRNA expression of TF on TFPI PAECs increased, similar to that on WT PAECs (Figure 6B).

Figure 6. Activation of PAECs by naïve and sensitized baboon sera to induce TF activity is inhibited by expression of TFPI.

Figure 6

Figure 6

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WT PAECs (white bars), TFPI PAECs (grey bars), and TFPI PAECs pretreated with anti-TFPI Ab at 50μg/ml for 30min (lined bars) were incubated with medium (control), 10% naïve (NBS), sensitized (SBS), or heat-inactivated sensitized (HI SBS) baboon serum for 8h. TF activity was determined by the recalcified clotting assay (A) and quantitative RT-PCR (B).

A. TFPI PAECs inhibited TF activity induced by all 3 baboon sera, and the effect was countered by the addition of anti-TFPI Ab. (# p<0.01 WT vs TFPI PAECs)

B. NBS, SBS or HI SBS increased the expression of TF mRNA in TFPI PAECs (*p<0.05, # p<0.01 vs control).

Atorvastatin inhibits the TF activity of PAECs induced by HI sensitized baboon serum

WT PAECs were pre-treated with atorvastatin with or without mevalonic acid for 16h before co-incubation with HI sensitized baboon serum. In the recalcified clotting assay, atorvastatin (at 10μM) inhibited TF activity induced on WT PAECs by HI sensitized baboon serum by about 50%. This was directly related to the effect of atorvastatin since the addition of mevalonic acid reversed its inhibitory effect (Figure 7A). Quantitative RT-PCR demonstrated that atorvastatin (at 10μM) inhibited the RNA expression of TF on WT PAECs induced by HI sensitized baboon serum (Figure 7B). The effect of atorvastatin was associated with dephosphorylation of Rho A and phosphorylation of Akt (Figure 7C).

Figure 7. Atorvastatin inhibits TF activity on PAECs induced by heat-inactivated sensitized baboon serum.

Figure 7

Figure 7

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WT PAECs were pretreated with atorvastatin (ATS) for 16h before incubation with heat-inactivated sensitized baboon serum (HI SBS). In one experiment, atorvastatin pretreatment was combined with mevalonic acid (MVA). TF activity was determined by the recalcified clotting assay (A) and quantitative RT-PCR (B). Rho A activation and Akt dephosphorylation were determined 8h after incubation with HI SBS (C).

A. Atorvastatin (at 10μM) inhibited 50% TF activity of PAECs after incubation with HI SBS. The effect was abolished by the addition of MVA.

B. After incubation with HI SBS, atorvastatin (at 10μM) inhibited the expression of TF mRNA. The effect was abolished by the addition of MVA (at 10 μM). (# p<0.01 compared to untreated control).

C. Atorvastatin led to dephosphorylation of Rho A and phosphorylation of Akt induced by HI SBS. The effect was reversed by the addition of MVA.

DISCUSSION

The expression of TF has been increasingly recognized as a factor in the development of hyperacute rejection and AHXR, and has been linked to the activation of PAECs. The resulting procoagulant stimuli have been hypothesized to play an important role in early xenograft failure. Previous studies by others have stressed that the activation of ECs as a trigger to thrombotic graft failure is dependent on the involvement of complement secondary to binding of xenoreactive Abs [16]. In a previous study from our group, antibody failed to activate PAECs without the involvement of complement [5]. Therefore, pigs expressing a complement-regulatory protein, e.g., CD46 or CD55, would be expected to protect ECs by regulating complement activation.

The results of the present study indicate that PAECs from pigs transgenic for CD46 are resistant to activation by naïve baboon serum, even in the presence of complement. These results correlate with previous data from our group that indicated that PAECs from GT-KO pigs additionally transgenic for CD46 were more resistant to primate complement-dependent cytotoxicity than those from GT-KO pigs alone [14].

By removing the major xenoreactive pig antigen (Gal), the generation of GT-KO pigs was initially expected to attenuate the primate humoral immune response and the subsequent AHXR. However, anti-nonGal Abs have emerged as an additional hurdle to successful xenotransplantation. Anti-nonGal Abs played a significant role in the rejection of organs from pigs transgenic for decay-accelerating factor in nonhuman primates [6]. In one previous study, the transplantation of kidneys from GT-KO pigs into baboons was associated with graft failure from severe AHXR; a marked induced Ab response to nonGal antigens mediated strong complement-dependent cytotoxicity against the GT-KO porcine target cells [6].

In the present study, HI sensitized baboon serum with a high titer of anti-nonGal Abs activated PAECs to express TF activity. PAECs that expressed CD46, that were resistant to activation by naïve baboon serum with low titers of anti-nonGal Abs, were activated by high-titer anti-nonGal Abs to express TF. Since the levels of total IgG were similar between naïve and sensitized baboons, we concluded that anti-nonGal Abs induce a procoagulant phenotype on PAECs independent of the presence of complement. This effect was related to IgG binding, but not to IgM binding. Our results confirmed the previous report that elicited Abs, potentially to nonGal epitopes, induce activation of PAECs and TF expression [4]. Similarly, Saethre et al. reported that human serum upregulated E-select in expression on Gal-depleted PAECs, which was largely complement-independent [17]. Importantly, natural anti-nonGal Abs had the same effect as elicited Abs if they were at high titer. Organs from GT-KO pigs, even if they express a human complement-regulatory protein, may be unable to prevent the development of a coagulopathy after transplantation into a primate.

This observation is mirrored in a recent large animal study [18]. After the transplantation of an organ from a GT-KO pig transgenic for a human complement-regulatory protein into a nonhuman primate, although the graft may remain functioning, the recipient may develop a consumptive coagulopathy, presumably associated with the procoagulant change on PAECs [18].

Since anti-nonGal Abs play an important role in the activation of vascular endothelial cells, attention has been directed to try to identify the nature of nonGal antigens. However, even if their structure(s) can be identified, if there are several such antigens, it may be a formidable task to further genetically modify GT-KO pigs to knockout these genes. Conventional systemic anticoagulant therapy, despite prolonging graft function, entails a significant risk of bleeding complications, which would be much less likely by the use of genetically-modified donor organs. Therefore, a more reasonable approach may be to generate GT-KO pigs that express an ‘anticoagulant’, or ‘anti-thrombotic’ gene, such as TFPI, to prevent the development of a procoagulant phenotype when PAECs are activated by anti-nonGal Abs. In a rodent model, Chen et al. reported that hearts from mice transgenic for a membrane-tethered fusion protein based on TFPI were resistant to humoral rejection after transplantation into rats. In contrast to WT mouse hearts, which were all rejected within 6 days, 100% of the hearts from the TFPI transgenic mice survived for >100 days when T cell-mediated rejection was inhibited [12].

Our current data demonstrate that TFPI-transgenic PAECs can inhibit TF activity on PAECs activated by fresh naïve baboon serum and HI sensitized baboon serum, even though there is an increase in TF mRNA after stimulation. One limitation of our study was that the effect of TFPI expression in the microvasculature, where thrombosis usually develops, was not examined. However, we would anticipate that a beneficial effect would still be observed. These promising results suggest that an anticoagulant transgene, such as TFPI, will be beneficial in overcoming the thrombotic microangiopathy that is associated with AHXR.

By inhibiting TF expression, statins have an anticoagulant effect that was first observed in monocytes and macrophages [19]. Our results demonstrate that, although atorvastatin almost completely suppressed the expression of TF mRNA, it only partially inhibited TF activity. The discrepancy may be because TF, once expressed on the cell surface, has a prolonged effect. This effect was observed at a higher concentration (10μM) than that of the usual peak plasma level (1μM) in humans after administration of atorvastatin [20]. This inhibitory effect on TF expression is compatible with the results of others [20] and is associated with the inhibition of rho-kinase-dependent Akt phosphorylation [15, 21]. Other benefits of the statins relevant to xenotransplantation have been reported. For example, atorvastatin inhibits the proliferative response of primate peripheral blood mononuclear cells and CD4+T cells when stimulated by PAECs [22]. The administration of atorvastatin in a nonhuman primate model of xenotransplantation would seem to be worthwhile. Although TFPI transgenic organs offer anti-thrombotic benefits to the graft, recent studies indicate that systemic coagulation disorders may persist [23]. Expression of TFPI on the endothelial cells alone may be insufficient. Other approaches, e.g., therapy with atorvastatin or other systemic agents, may be necessary to completely overcome this challenge.

In summary, anti-nonGal IgG Abs activated PAECs to induce TF activity through a complement-independent pathway. This implies that GT-KO pigs expressing a complement-regulatory protein may be insufficient to prevent the development of immune-dependent procoagulant changes on PAECs. The effect can be inhibited by different mechanisms, e.g., by the expression of an ‘anticoagulant’ gene (TFPI) on the PAECs or by systemic therapy with atorvastatin. Our results suggest potential approaches to resolve the problem of thrombotic microangiopathy and consumptive coagulopathy after xenotransplantation.

Acknowledgments

Work on xenotransplantation in the Thomas E. Starzl Transplantation Institute of the University of Pittsburgh is supported in part by NIH grants # U01 AI068642 and # R21 A1074844, and by Sponsored Research Agreements between the University of Pittsburgh and Revivicor, Inc., Blacksburg, VA.

FUNDING SOURCES

Work in our laboratory is supported in part by NIH grants U01-AI68642 and R21-AI074844-01 and by Sponsored Research Agreements between the University of Pittsburgh and Revivicor, Inc., Blacksburg, VA, USA.

ABBREVIATIONS

Abs

antibodies

AHXR

acute humoral xenograft rejection

FVII/VIIa

Factor VII/Factor VIIa

Gal

Galα1,3Gal

GT-KO

α1,3-galactosyltransferase gene-knockout

HI

heat-inactivated

PAECs

porcine aortic endothelial cells

TF

tissue factor

TFPI

tissue factor pathway inhibitor

WT

wild-type

References

  • 1.Cooper DK, Gollackner B, Sachs DH. Will the pig solve the transplantation backlog? Annu Rev Med. 2002;53:133–47. doi: 10.1146/annurev.med.53.082901.103900. [DOI] [PubMed] [Google Scholar]
  • 2.Kuwaki K, Tseng YL, Dor FJ, Shimizu A, Houser SL, Sanderson TM, Lancos CJ, Prabharasuth DD, Cheng J, Moran K, Hisashi Y, Mueller N, Yamada K, Greenstein JL, Hawley RJ, Patience C, Awwad M, Fishman JA, Robson SC, Schuurman HJ, Sachs DH, Cooper DK. Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat Med. 2005;11:29–31. doi: 10.1038/nm1171. [DOI] [PubMed] [Google Scholar]
  • 3.Nagayasu T, Saadi S, Holzknecht RA, Plummer TB, Platt JL. Expression of tissue factor mRNA in cardiac xenografts: clues to the pathogenesis of acute vascular rejection. Transplantation. 2000;69:475–82. doi: 10.1097/00007890-200002270-00003. [DOI] [PubMed] [Google Scholar]
  • 4.Gollackner B, Goh SK, Qawi I, Buhler L, Knosalla C, Daniel S, Kaczmarek E, Awwad M, Cooper DK, Robson SC. Acute vascular rejection of xenografts: roles of natural and elicited xenoreactive antibodies in activation of vascular endothelial cells and induction of procoagulant activity. Transplantation. 2004;77:1735–41. doi: 10.1097/01.tp.0000131167.21930.b8. [DOI] [PubMed] [Google Scholar]
  • 5.Lin CC, Chen D, McVey JH, Cooper DK, Dorling A. Expression of Tissue Factor and Initiation of Clotting by Human Platelets and Monocytes After Incubation With Porcine Endothelial Cells. Transplantation. 2008;86:702–9. doi: 10.1097/TP.0b013e31818410a3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Chen G, Qian H, Starzl T, Sun H, Garcia B, Wang X, Wise Y, Liu Y, Xiang Y, Copeman L, Liu W, Jevnikar A, Wall W, Cooper DK, Murase N, Dai Y, Wang W, Xiong Y, White DJ, Zhong R. Acute rejection is associated with antibodies to non-Gal antigens in baboons using Gal-knockout pig kidneys. Nat Med. 2005;11:1295–8. doi: 10.1038/nm1330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Chen D, Riesbeck K, McVey JH, Kemball-Cook G, Tuddenham EG, Lechler RI, Dorling A. Regulated inhibition of coagulation by porcine endothelial cells expressing P-selectin-tagged hirudin and tissue factor pathway inhibitor fusion proteins. Transplantation. 1999;68:832–9. doi: 10.1097/00007890-199909270-00016. [DOI] [PubMed] [Google Scholar]
  • 8.LaRosa JC. Pleiotropic effects of statins and their clinical significance. Am J Cardiol. 2001;88:291–3. doi: 10.1016/s0002-9149(01)01643-5. S0002-9149(01)01643–5. [DOI] [PubMed] [Google Scholar]
  • 9.Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen SH, Ball S, Specht SM, Polejaeva IA, Monahan JA, Jobst PM, Sharma SB, Lamborn AE, Garst AS, Moore M, Demetris AJ, Rudert WA, Bottino R, Bertera S, Trucco M, Starzl TE, Dai Y, Ayares DL. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science. 2003;299:411–4. doi: 10.1126/science.1078942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Loveland BE, Johnstone RW, Russell SM, Thorley BR, McKenzie IF. Different membrane cofactor protein (CD46) isoforms protect transfected cells against antibody and complement mediated lysis. Transpl Immunol. 1993;1:101–8. doi: 10.1016/0966-3274(93)90002-p. [DOI] [PubMed] [Google Scholar]
  • 11.Riesbeck K, Dorling A, Kemball-Cook G, McVey JH, Jones M, Tuddenham EG, Lechler RI. Human tissue factor pathway inhibitor fused to CD4 binds both FXa and TF/FVIIa at the cell surface. Thromb Haemost. 1997;78:1488–94. [PubMed] [Google Scholar]
  • 12.Chen D, Weber M, McVey JH, Kemball-Cook G, Tuddenham EG, Lechler RI, Dorling A. Complete inhibition of acute humoral rejection using regulated expression of membrane-tethered anticoagulants on xenograft endothelium. Am J Transplant. 2004;4:1958–63. doi: 10.1111/j.1600-6143.2004.00625.x. [DOI] [PubMed] [Google Scholar]
  • 13.Ayares D, Phelps CJ, Vaught TD, Ball S. Genetic engineering of pigs for improved transplant outcomes. Xenotransplantation. 2007;14:428. [Google Scholar]
  • 14.Hara H, Long C, Lin YJ, Tai HC, Ezzelarab M, Ayares D, Cooper DK. In vitro investigation of pig cells for resistance to human antibody-mediated rejection. Transpl Int. 2008;21:1163–74. doi: 10.1111/j.1432-2277.2008.00736.x. [DOI] [PubMed] [Google Scholar]
  • 15.Eto M, Kozai T, Cosentino F, Joch H, Luscher TF. Statin prevents tissue factor expression in human endothelial cells: role of Rho/Rho-kinase and Akt pathways. Circulation. 2002;105:1756–9. doi: 10.1161/01.cir.0000015465.73933.3b. [DOI] [PubMed] [Google Scholar]
  • 16.Yang YG, Sykes M. Xenotransplantation: current status and a perspective on the future. Nat Rev Immunol. 2007;7:519–31. doi: 10.1038/nri2099. [DOI] [PubMed] [Google Scholar]
  • 17.Saethre M, Baumann BC, Fung M, Seebach JD, Mollnes TE. Characterization of natural human anti-non-gal antibodies and their effect on activation of porcine gal-deficient endothelial cells. Transplantation. 2007;84:244–50. doi: 10.1097/01.tp.0000268815.90675.d5. [DOI] [PubMed] [Google Scholar]
  • 18.Kelishadi SS, Zhang T, Stoddard T, Azimzadeh AM, Welty E, Avon CJ, Larris A, Cheng XF, Barth R, Pierson RN., 3rd Addition of HDAF to the Gal -KO background improved survival and function of pig kidneys in baboons. Am J Transplant. 2008;8:205. [Google Scholar]
  • 19.Aikawa M, Rabkin E, Sugiyama S, Voglic SJ, Fukumoto Y, Furukawa Y, Shiomi M, Schoen FJ, Libby P. An HMG-CoA reductase inhibitor, cerivastatin, suppresses growth of macrophages expressing matrix metalloproteinases and tissue factor in vivo and in vitro. Circulation. 2001;103:276–83. doi: 10.1161/01.cir.103.2.276. [DOI] [PubMed] [Google Scholar]
  • 20.Davidson MH, Stein EA, Dujovne CA, Hunninghake DB, Weiss SR, Knopp RH, Illingworth DR, Mitchel YB, Melino MR, Zupkis RV, Dobrinska MR, Amin RD, Tobert JA. The efficacy and six-week tolerability of simvastatin 80 and 160 mg/day. Am J Cardiol. 1997;79:38–42. doi: 10.1016/s0002-9149(96)00742-4. [DOI] [PubMed] [Google Scholar]
  • 21.Ferrara DE, Swerlick R, Casper K, Meroni PL, Vega-Ostertag ME, Harris EN, Pierangeli SS. Fluvastatin inhibits up-regulation of tissue factor expression by antiphospholipid antibodies on endothelial cells. J Thromb Haemost. 2004;2:1558–63. doi: 10.1111/j.1538-7836.2004.00896.x. [DOI] [PubMed] [Google Scholar]
  • 22.Ezzelarab M, Welchons D, Torres C, Hara H, Long C, Yeh P, Ayares D, Cooper DK. Atorvastatin down-regulates the primate cellular response to porcine aortic endothelial cells in vitro. Transplantation. 2008;86:733–7. doi: 10.1097/TP.0b013e3181821cad. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Lin CC, Cooper DK, Dorling A. Coagulation dysregulation as a barrier to xenotransplantation in the primate. Transpl Immunol. 2009;21:75–80. doi: 10.1016/j.trim.2008.10.008. [DOI] [PMC free article] [PubMed] [Google Scholar]

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