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Cardiovascular Research 2007 73(1):143-152; doi:10.1016/j.cardiores.2006.10.027
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Copyright © 2006, European Society of Cardiology

Reduction of hyperacute rejection and protection of metabolism and function in hearts of human decay accelerating factor (hDAF)-expressing pigs

Ryszard T. Smolenskia,d, Monica Fornib, Massimo Maccherinic, Maria Laura Baccib, Ewa M. Slominskad, Hongjun Wange,1, PierMaria Fornasarif, Roberto Giovannonie, Felicetta Simeonec, Augusta Zannonib, Giacomo Fratig, Ken Suzukia, Magdi H. Yacouba and Marialuisa Lavitranoe,*

aHeart Science Centre, Imperial College at Harefield Hospital, Harefield, Middlesex, UB9 6JH, U.K.
bDIMORFIPA, University of Bologna, 40064 Bologna, Italy
cIst.Chir.Tor. Card. University of Siena, Italy
dDepartment of Biochemistry, Medical University of Gdansk, 80-211 Gdansk, Debinki 1, Poland
eDepartment of Surgical Sciences, University of Milano-Bicocca, 20052 Milano, Italy
fBlood Transfusion Service, Rizzoli Orthopaedic Institute, Bologna, Italy
gUniversity "La Sapienza", 00161 Roma, Italy

* Corresponding author. Dept. of Surgical Sciences, Università di Milano-Bicocca, Via Cadore, 48, 20052 Monza, Milano, Italy. Tel.: +39 02 6448 8336; fax: +39 02 6448 8341. Email address: marialuisa.lavitrano{at}unimib.it

Received 28 July 2006; revised 21 October 2006; accepted 26 October 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The use of pig hearts can solve the problem of shortage of donor hearts for transplantation. However, targeting rejection by single genetic modification was proven to be ineffective, highlighting the requirement for complex genetic modifications and more effective methods for transgenic animal production. We evaluated here whether hearts of hDAF transgenic pigs generated using our technique sperm-mediated gene transfer (SMGT) will be protected from structural damage, metabolic changes, and mechanical dysfunction during perfusion with human blood.

Methods: Hearts from control (C, n=6) or transgenic (T, n=5) pigs were perfused ex vivo for 4 h with fresh human blood using the ex vivo working mode system allowing monitoring of the function, metabolism, and structure.

Results: Cardiac output (mean±SEM) was maintained in T constant throughout the experiment, at 3.58±0.36 and 3.83±0.14 l/min after 30 min and 4 h, respectively, while cardiac output decreased to 1.95±0.35 l/min in C after 30 min of perfusion (p<0.01 vs. T). The maximum increase in coronary perfusion pressure was reduced in T to 154±16% as compared to C (237±10%, p<0.001). Myocardial ATP after 4 h was 21.1±1.1 nmol/mg dry wt (similar to initial) in T, while it decreased in C to 17.2±1.4 (p<0.05). Deposition of complement factors C3 and C5b9 was present in C but not in T after perfusion.

Conclusion: We have shown that hearts from hDAF transgenic pigs produced by SMGT are protected during perfusion with human blood and are metabolically stable and maintain mechanical function above the threshold level for life support.

KEYWORDS Xenotransplantation; Transgenic pigs; Hyperacute rejection; SMGT; Cardiac function

Abbreviations: BSA, bovine serum albumine • hDAF, human decay accelerating factor • LDTI, low-density triton-insoluble • MoAb, monoclonal antibody • NK cells, natural killer cells • SFM, swine fertilization medium • SMGT, sperm mediated gene transfer.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Heart failure is currently one of the major causes of mortality and factors affecting quality of life in humans. Despite advances in pharmacological treatment and recent developments in mechanical circulatory support devices, cardiac transplantation remains the most effective therapy of end-stage heart failure. However, availability of human donor hearts is limited and only a small percent of those who would benefit from this treatment could actually be transplanted. It is expected that the disparity between organ availability and demand will increase even further in the future. One possible solution of the donor organ shortage is the use of animal organs [1,2]. To date pigs seem to be the best source of organs for xenotransplantation because of their physiological compatibility and ethical acceptability. Other practical aspects such as low costs, short breeding cycle, multiple offspring and possibility to obtain specific pathogen-free colonies are also important [1–4]. However, following transplantation into primates, pig hearts undergo a rapid and vigorous reaction termed hyperacute rejection causing total dysfunction within minutes. This reaction is triggered by IgM antibodies binding to gal-{alpha}-gal epitopes. This is followed by complement activation with contribution of the other mechanisms such as platelet aggregation, coagulation as well as T lymphocyte, NK cells and neutrophil-mediated cytotoxic effects [1,5–8].

Identification of the major mechanisms involved in hyperacute rejection following xenotransplantation indicated that molecular interventions such as transgenic expression of human complement regulatory proteins on the surface of pig organs or knock-out of the genes responsible for {alpha}-gal epitope production would exert a protective effect. The high efficiency of these strategies in experimental studies revived hopes for its effective application in humans [9–12]. Despite some concerns about possible risks of transfer of the animal retroviruses into humans, there is a growing consensus that preclinical studies should be performed to test possible solutions to rejection and infectious risks rather than abandoning this life-saving opportunity [13–15].

To date, a major step forward in the field of xenotransplantation came with the generation of alpha-1,3-galactosyltransferase knockout pigs [16,17]. Recently the use of the heart and the kidney from these pigs showed an impressive extension of the survival when transplanted in non-human primates [18,19]. A successful approach to inhibit xenograft rejection in pig to primate models has been also inhibiting complement activation, with the expression of human complement inhibitors on cell surfaces or with the depletion of the xenoreactive natural antibodies. Transgenic pigs for human decay accelerating factor (hDAF) have been generated by a number of groups, and prolonged survival of transgenic pig organs was observed following transplantation into primates. However, it appears that production of multi-gene engineered pigs will be essential, since expression of hDAF on its own or other single gene approaches does not sufficiently protects pig tissues from rejection [20,21]. An important challenge is therefore the development of effective and inexpensive procedures for transgenic animal generation. Sperm-mediated gene transfer (SMGT) developed by us several years ago [22] appears to be an efficient and cost-effective procedure for that purpose [23]. We successfully established a pig strain expressing human decay accelerating factor (hDAF) [24]. We have produced 34 lines of transgenic pigs expressing human DAF. We showed expression of hDAF in different cells, tissues and organs such as the heart, liver, lung, kidney, aorta, and PBMCs. The highest expression of hDAF was demonstrated in the vascular endothelium by histological analysis of tissue sections. hDAF expression in macrophages from 16 different transgenic lines of pigs resulted in resistance to complement-mediated lysis when challenged with human serum [24].

An important issue is the development and application of methods for testing potential xenografts. Since life-supporting transplantation into primates is not applicable on a routine basis, a number of tests ranging from in vitro cultured cell models to ex vivo organ perfusion have been developed.

In this study we applied newly developed ex vivo perfusion system (Fig. 1) to perform three functions: 1. to functionally characterize hearts of transgenic pigs expressing hDAF generated by the SMGT method during perfusion with human blood, 2. to evaluate whether hearts from those pigs will be protected against hyperacute rejection, 3. to evaluate how this protection will relate to mechanical, vascular and metabolic function.


Figure 1
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  		Fig. 1 Schematic diagram of the system for perfusion of pig hearts with human blood. Black marks indicate coronary perfusion circuit. Gray marks indicate functional assessment circuit. Arrows indicate direction of flow. The coronary perfusion side included a centrifugal pump, a blood reservoir, a thermostatically controlled pediatric blood oxygenator with outflow connected to aortic cannula, a water jacketed heart compartment, a hemofilter and thermostated (37 °C) circulator. The functional assessment side consisted of a left ventricular balloon connected to Y shaped tube fitted with inlet and outlet valves. The inlet tube was connected to a vessel maintaining constant filling pressure at 15 mm Hg. The outlet tube was fitted with electromagnetic flow probe and was opened to air at the height of 65 mm Hg.

 

   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Generation of transgenic pigs and assessment of hDAF expression in transgenic pig hearts
All experiments were performed in compliance with European, national and institutional regulations on animal experimentation and genetic manipulation. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Transgenic pigs expressing hDAF were generated by introducing hDAF minigene into sperm cells followed by artificial insemination as described in detail previously [23,24].

Ten hDAF transgenic founders were bred with wild type pigs to produce F1 progeny. hDAF transgene integration and expression were studied at birth. Genomic DNA and RNA from tail and ear samples were analyzed by Southern blot, PCR, RT-PCR and Northern blot according to standard protocols. hDAF protein expression was studied in umbilical cord sections by immunohistochemistry (see Ref. [24] for details). Five hDAF transgenic pigs (F1 progeny, 3 piglets from high expresser founder A01 and 2 from low expresser founder D07 [24]) were selected at birth on the basis of positivity for hDAF RNA expression and then used in the ex vivo heart perfusion experiments herein described. The 3 high expressing pigs (A01-223, A01-226, A01-227) had fivefold more hDAF mRNA on average than the 2 low expressing pig (D07-411, D07-414). The intensity of hDAF transcript band in Northern blot analysis was quantified using Molecular Image FX Documentation System (Bio-Rad Laboratories, Milan, Italy). hDAF expression was also confirmed at RNA and protein level in heart samples from the selected pigs. RNA and protein extracts were prepared from frozen biopsies and analyzed by RT-PCR and Western blot, respectively as previously described [24]. Low-Density Triton-Insoluble (LDTI) fraction V, containing the bulk of the hDAF in the transgenic pig hearts, was recovered and Western blotted using IA10 anti-hDAF MoAb (BD Pharmingen, Milan, Italy) at 1:500 dilution as previously described [24].

2.2. The perfusion system
The perfusion system was designed to allow maintenance of constant coronary flow of perfusing blood and simultaneous assessment of cardiac function under physiological preload and afterload. Fig. 1 presents the outline of the system. The coronary perfusion side included a centrifugal pump (Medtronics), a blood reservoir, a thermostatically controlled pediatric blood oxygenator with outflow connected to aortic cannula, a water jacketed heart compartment, a hemofilter continuously flushed with a Krebs–Henseleit buffer prepared as described before [25], a thermostated circulator maintaining the temperature of the blood oxygenator and heart compartment at 37 °C. The functional assessment side consisted of a working balloon made of PVC cling-film connected to a 2 cm diameter Y shaped tube fitted with inlet and outlet flap-type valves. The inlet tube was connected to a vessel maintaining constant filling pressure at 15 mm Hg. The outlet tube was opened to air at the height of 65 mm Hg. Ejected fluid was then collected and returned to the system. The circuit for function assessment was filled with saline. Functional parameters were continuously recorded by a computer-assisted system (AcqKnowledge, Linton Instruments, U.K) that continuously acquired data from pressure transducers located at the outlet from left ventricle and in the aortic root and electromagnetic flow meter (Nihon-Kohden, Japan) measuring flow in the aortic outflow.

2.3. Surgical procedure for heart collection
Transgenic (T, n=5) and Control (C, n=6) pigs of either sex weighing about 106.4±7.3 kg (mean±SEM), were utilized. The pigs were sedated with an injection of azaperone (3 mg/kg) (Stresnil®, Janssen-Cilag SpA, Milan, Italy) and atropine sulphate (0.02 mg/kg) (Atropina Solfato®, A. Fatro SpA, Bologna, Italy) and anaesthesia was induced with intramuscular ketamine (20 mg/kg) (Ketavet 100®, Intervet Italia Srl). After the onset of anaesthesia, pigs were intubated and muscle relaxation was obtained with intravenous infusion of pancuronium bromide (0.1 mg/kg) (Organon Teknika B.V. Boxtel, Nederland). Ventilation was controlled with a Servo ventilator (Siare srl Hospital Supplies, Bologna, Italy). Anaesthesia was maintained with isofluorane 1.5 to 2% (Forane, Abbott Laboratories–North Chicago, IL). The chest was then opened by median sternotomy. After systemic heparinization (500 IU/kg) the ascending aorta was cannulated with a 9-gauge aortic root cannula (Medtronic Italia SpA, Milano, Italy) and clamped. Before removal from the chest, hearts were then infused with 1 l of ice-cold St. Thomas' Hospital No.1 cardioplegic solution flushed in the aortic root at a pressure of 60 mm Hg. The appendices of the left and right atrium were opened to keep the heart decompressed, and saline ice-slush (0.5 °C) was placed around the heart. Aorta was prepared and cannula introduced and fixed followed by introduction of the suture around mitral valve annulus for fixing balloon cannula. Hearts were then connected to perfusion apparatus by attaching aortic cannula to outflow tubing from oxygenator. Care was taken to avoid introduction of air by maintaining slow flow during cannulation. Following cannulation and start of perfusion, balloon catheter for functional assessment was introduced via mitral valve. To prevent slipping of the balloon catheter part was secured with strong suture around mitral annulus.

2.4. Ex vivo perfusion of pig hearts with human blood
Before cannulation, the system was first primed with saline and then filled with 1.2 l of freshly collected heparinized (12000 U) human blood pooled from three haemocompatible donors of A or O blood types, tested positive for complement activation.

Blood pH and calcium concentration were adjusted before connection of the heart with NaHCO3 and CaCl2 solutions and Insulin (2 I.U.) was added to the blood. Following connection of the heart through the aorta, the initial flow was adjusted to obtain a perfusion pressure of 60 mm Hg. This was achieved with a blood flow rate within the range of 0.4–0.6 l/min in all experiments. This flow setting was maintained throughout perfusion. Hearts were defibrillated at the start of perfusion. All perfusion experiments were carried out for 4 h. Hearts were weighted immediately after the explant and after 4 h of perfusion.

2.5. Histological analysis
Histological analyses were performed on biopsies collected at the beginning of the experiment and just before hearts were disconnected from the system. Specimens were fixed with formaldehyde, paraffin-embedded and stained with hematoxylin/eosin according to standard procedures. Edema, thrombosis, endothelial damage and necrosis were evaluated by semiquantitative analyses performed by 3 independent observers based on the tissue percentage involved in the pathological processes on 10 high power fields (HPF, x40): 0, absent; 1, less than 33%; 2, 34–66%; 3, more than 67%.

2.6. Immunohistochemistry
Immunohistochemical studies were carried out on frozen tissue sections (7 µm) fixed with 2% (wt/vol) paraformaldehyde / 1% (vol/vol) acetic acid in PBS for 10 min at RT. To evaluate hDAF expression, slides were incubated with IF-7 anti-hDAF MoAb (Upstate Biotechnology, Lake Placid, NY) at 1:50 dilution (for details see [24]). To evaluate complement deposition slides were incubated with anti-C3 and anti-C5b9 MoAbs (DAKO, Carpinteria CA) after being washed with universal biotinylated anti-rabbit, anti-mouse immunoglobulins (DAKO LSAB 2 Kit, Peroxidase Universal, DAKO). Detection was performed using streptavidin-conjugated peroxidase (DAKO). Staining was revealed using 3-amino-9-ethylcarbazole (AEC, DAKO) as a colorimetric substrate. Aqueous hematoxylin was used as a counterstain. Sections were mounted in an aqueous mounting medium and analyzed by Zeiss light microscopy. The intensity of staining was graded independently by 3 observers using the following criteria: 0, absent; 1, faint; 2, moderate; 3, intense; 4, very intense.

2.7. Determination of ATP and related metabolites in cardiac biopsies
Biopsies for evaluation of nucleotide concentrations were collected using Travenol Tru-Cut biopsy needles. The biopsies were frozen in liquid nitrogen immediately (within 3 s) after collection. They were then transferred into cryo-vials under liquid nitrogen, freeze dried and extracted with 0.4 M perchloric acid. Neutralized extracts were analyzed with high performance liquid chromatography (HPLC) according to our reverted-phase or ion-exchange procedures described in detail previously [26–28].

2.8. Statistical analysis
All values are presented as means±standard error of the mean (S.E.M.). Statistical analyses of the differences in cardiac output, dP/dt, perfusion pressure and heart weight were performed using Students t-test. Metabolic differences were compared using a one-way analysis of variance followed by Student–Newmann–Keuls test. Morphological and immunohistochemical data were compared using the non-parametric Mann–Whitney-U Test. Differences were considered significant at p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Expression of hDAF in hearts of transgenic pigs
Three hDAF transgenic pigs obtained by breeding founder A01 and two obtained by breeding founder D07 with wild type pigs [24], were selected for perfusion experiments. mRNA expression was analyzed by Northern blot. In F1 piglets mRNA levels were found to be similar to the corresponding founder (data not shown). Expression of hDAF was confirmed in transgenic pig hearts biopsies collected at the beginning of the perfusion experiments. Total RNA was prepared from snap-frozen biopsies and analyzed by RT-PCR. The specific primers used amplified a 1059 bp fragment, corresponding to the hDAF full transcript (Fig. 2A). Western blot on heart lysates from the 5 transgenic pigs was tested for expression of hDAF protein. We previously described that hDAF transgenic protein present in pig cells was located in caveolae, the low-density Triton-insoluble (LDTI) plasma-membrane domains of cell surface, similarly to DAF in human cells [24]. Western blot on heart lysates from the 5 transgenic pigs was tested for expression of hDAF protein. Protein analysis of fraction V of LDTI preparations from heart lysates demonstrated that hDAF was present and migrated similarly as a unique band of 66 kDa in samples from the 5 transgenic pigs selected for perfusion experiments and from human heart (Fig. 2B). Expression of hDAF was also shown by immunohistochemistry by using an anti-hDAF MoAb (Fig. 2C and Table 1). Hearts from control pigs tested negative for hDAF RNA and protein expression.


Figure 2
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  		Fig. 2 Expression of hDAF in transgenic pigs used for ex vivo heart perfusion with human blood. A) RT-PCR analysis showed one band of 1059 bp in RNA extracted from heart of the 5 transgenic pigs (lanes 2–6); no bands were observed in controls (lane 1). hDAF primers were used to amplify cDNA obtained by reverse transcription of 1 µg of total RNA. RT-PCR products were analyzed on agarose gel followed by Southern blot. The RT-PCR experiments were subjected to routine controls. The primers used amplified fragments of 1575 and 1059 bp from the hDAF minigene and the cDNA, respectively and discriminated between amplification of transgenic DNA or of cDNA on the basis of expected size. The size of the fragments obtained by RT-PCR was evaluated with respect to 1 Kbp ladder (not shown). B) Western Blot analysis of fraction V from Low-Density Triton-Insoluble protein extract. The LDTI domains were isolated from human, transgenic pig and control pig hearts by sucrose-density centrifugation and analyzed on 10% SDS-PAGE under non-reducing conditions, blotted and probed with IA10 anti-hDAF monoclonal antibody, which recognizes a specific band of 66 kDa. Lane 1: human sample; lane 2: high expresser hDAF transgenic pig A01-223; lane 6: low expresser hDAF transgenic pig D07-411. No specific bands were revealed by the antibody in the control pig (lane 4), as well as in non-transgenic siblings (lanes 3 and 5) indicating that the antibody does not cross-react with pig DAF. C) Immunostaining for hDAF on frozen tissue sections from transgenic pig A01-223. Sections of hearts from transgenic (central panel) pig were stained with IF-7 anti-hDAF monoclonal antibody or an isotype matched control (left panel). The heart shows hDAF expression mostly in the plasmalemma of the muscle fibers. Sections of heart from a control pig were stained with the same anti-hDAF monoclonal antibody (right panel). Staining was with the ABC method with hematoxylin counterstaining. Magnification is 40x.

 

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  		Table 1 Semiquantitative analyses of tissue biopsies from transgenic (A01-223, A01-226, A01-227, D07-411, D07-414) and control (C01-C06) pig hearts

 
3.2. Cardiac function and coronary perfusion pressure during ex vivo perfusion of transgenic or control pig hearts with human blood
All hearts in both transgenic and control groups continue to beat for the entire duration of the experiment. Cardiac volume output was stable in transgenic pig hearts while in controls, function initially decreased to about 50% of that observed in transgenic hearts and then slightly recovered but remained significantly lower than in transgenic hearts (Fig. 3A). Maximal rate of left ventricular pressure increase (+dP/dt, Fig. 3B) followed similar pattern as have maximal rate of left ventricular pressure decrease (–dP/dt, Fig. 3C). This difference after 30 min was the most prominent (3x lower values in control hearts than in transgenic). Interestingly, substantial recovery was observed after 4 h of perfusion in control hearts. There were no differences in heart rate between control or transgenic hearts at any time point of perfusion (Fig. 3D). Coronary perfusion pressure (Fig. 3E) increased in control hearts to 237±10% of initial at its peak at 1 h of perfusion while in transgenic hearts the increase in perfusion pressure was reduced to 154±16% (p<0.001).


Figure 3
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  		Fig. 3 A) Cardiac output, B) maximal rate of left ventricular pressure increase (+dP/dt max), C) maximal rate of left ventricular pressure decrease (–dP/dt max) and D) heart rate after 30 min and 4 h of perfusion in transgenic for hDAF or control pig hearts perfused with human blood using the system shown in Fig. 1. Values are mean±S.E.M. *p<0.01 vs. Control, #p<0.05 vs. Control. E) Coronary pressure during perfusion of transgenic and control hearts with human blood using the system shown in Fig. 1. Values are mean±S.E.M. *p<0.05 vs. Control.

 
3.3. Morphology of transgenic and control hearts after perfusion with human blood
Heart weight increased after 4 h of perfusion with human blood in controls by 39.7%±3.5 while increase in transgenics was 21.0%±3.6 (p<0.005). Fig. 4 presents the microscopic structure of the transgenic and control hearts after perfusion. Semiquantitative analyses of morphological data are summarized in Table 1. Pericellular edema was negligible in transgenics (score 1 for all hearts) while cardiac fibers in controls were edematous (score range 2–3; p<0.005) and showed diffuse vacuolization. The endothelium status was normal in transgenics (score range 0–1), while in controls the endothelial lamina was discontinuous (score range 2–3; p<0.005). Furthermore a moderate thrombosis was observed in controls (score range 1–2) and not in transgenics (0–1; p<0.05) while necrosis was an uncommon feature. At ultra-structural level, mitochondria and edema filled vacuolar space in controls contrasting transgenics that showed mitochondria in good shape (data not shown). Analysis of complement deposition in pig hearts perfused with human blood indicated that C3 was abundantly deposited in control pig hearts (score range 2–3), while transgenics showed a sparse presence of C3 (score range 1–2; p<0.01) and no C5b9 deposition was found in transgenic pig hearts (score 0 for all hearts) while in controls was abundant (score range 2–3; p<0.005) (Table 1, Fig. 5).


Figure 4
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  		Fig. 4 Microscopic analysis of left ventricular specimens collected from hDAF transgenic A01-223 and control hearts at the end of 4 h perfusion with human blood. Slides were stained with hematoxylin/eosin. Magnification is 40x.

 

Figure 5
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  		Fig. 5 Deposition of C3 and C5b9 in hDAF transgenic A01-223 and control pig hearts at the end of 4h perfusion with human blood. Magnification is 40x.

 
3.4. Cardiac ATP concentration following perfusion of pig hearts with human blood
Myocardial ATP concentration was maintained at its initial level in transgenic pig hearts following 4 h of perfusion with human blood. In control pig hearts, however, ATP concentration decreased slightly but significantly by about 15% as compared to initial and transgenic hearts (Fig. 6A). Concentration of NAD was not affected (Fig. 6A). No differences in phosphocreatine or creatine concentrations or their ratio were observed. Phosphocreatine concentration was 41.1±4.7 and 44.7±3.8 nmol/mg dry wt in control and transgenic hearts, respectively, after 4 h of perfusion while respective creatine concentrations were 21.4±2.7 and 20.5±2.5 nmol/mg dry wt. However, an interesting change in UTP concentration was noted. An approximately two-fold increase in UTP concentration (expressed as % of ATP) was observed in both transgenic and control hearts (Fig. 6B).


Figure 6
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  		Fig. 6 Myocardial ATP and NAD concentration (A) and GTP, UTP and CTP concentrations (B) in control and hDAF transgenic hearts at the beginning and the end of 4 h perfusion with human blood. Values are mean±S.E.M. *p<0.05 vs. 0 min, #p<0.05 vs. Control.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
This study demonstrated that hearts of hDAF transgenic pigs generated using SMGT are protected from hyperacute rejection when perfused with human blood as indicated by functional and molecular analysis and that at physiological preload and afterload transgenic hearts maintain cardiac output adequate for life support. It must be pointed out that similar results were obtained among individual experiments using hearts exhibiting low hDAF RNA and protein expression or high hDAF RNA and protein expression. Possibly this could be due because the levels of expression of hDAF protein in our transgenic hDAF low expresser pigs were anyhow comparable to the levels in human heart as demonstrated in Western blot experiments showed in Fig. 2B.

This study also demonstrated advantages of using an ex vivo human blood perfusion system for studying function, biochemistry and immune mechanisms in transgenic hearts under conditions closely resembling clinical situations in the first hours post xenotransplantation.

Although hearts in both groups sustained beating for the duration of the experiment, coronary perfusion pressure increased much less in transgenic animals. This may indicate reduced edema resulting from activation of complement and inflammatory mechanisms in hearts expressing hDAF or better protection vascular tone regulation mechanisms. Attenuation of hyperacute rejection was further confirmed by microscopic analysis of cardiac specimens. There was no structural damage in transgenic hearts. Deposition of C3 complement factor was markedly attenuated in transgenic hearts and no trace of C5b9 was found. It must be pointed out however, that rejection related changes were not entirely blocked in transgenic hearts. This could be related to the fact that, in the ex vivo perfusion of pig hearts with human blood, blood-group types A and O were used to maximize rejection damage that is apparently reduced with B and AB blood-group types [29]. Some damage still observed in transgenic hearts could be a consequence of the decreased activity of ecto-5'-nucleotidase. We recently demonstrated a novel potential mechanism of xenograft dysfunction that may involve a reduction in the capacity of the endothelium to convert pro-inflammatory and pro-aggregatory extracellular nucleotides into adenosine due to a specific decrease of ecto-5'-nucleotidase activity that was observed despite expression of hDAF following contact with human blood [30]. These results suggest that complement inhibition by hDAF expression is not sufficient to fully protect porcine organs from injury from human blood.

Although it has been previously established that the expression of human DAF effectively attenuates hyperacute rejection in both ex vivo and in vivo models [11,31], procedures used to generate these transgenic animals were complicated, expensive and inefficient. Demonstration of equivalent results with SMGT indicates that eventual optimization of efficiency and possibility of multiple candidate genes expression, which may protect against rejection, could become more accessible [21].

Our application of a novel design of perfusion system represents another major development presented in this paper. With the system described, stable physiological cardiac output has been achieved for the duration of 4 h. This contrasts previous ex vivo systems used for perfusion of pig hearts with human blood where physiological output has been achieved only initially and then a gradual decrease has been observed to almost zero after 4–6 h even in transgenic hearts [11,32–34]. The separation of working side and coronary perfusion side applied here has been important in reducing the volume of human blood necessary to conduct experiments and to decrease the blood trauma inevitable in standard working-mode perfusion systems. The system presented here allows better control of both coronary perfusion as well as preload and afterload of the heart. Although it might be said that constant flow coronary perfusion is not compatible with physiological situations it is sometimes used clinically during cardiac surgery. Our data shows that after the initial rise in perfusion pressure with its peak after 60 min, a gradual decrease and return to initial values have been observed, even in non-transgenic hearts. We suggest therefore that direct coronary perfusion in a flow controlled mode rather than in pressure controlled mode could be the right approach in a clinical trial for xenotransplantation.

There are several limitations of the system used in this study. First, it allows for only several hours of perfusion essentially limiting its application to study hyperacute rejection. Secondly, not all mechanisms of rejection could be reproduced such as platelet mediated that was blunted by high dose heparin. Ex vivo system also neglects effect of other organs on blood homeostasis. Despite efforts to make close match to physiological conditions, working balloon used in this study may not accurately reproduce mechanical function in vivo. However, its limitations such as increased flow restriction due to lower diameter of tubing than natural flow path could lead only to underestimation of cardiac output. This may explain why maximum values of cardiac output were slightly below what is expected for cardiac size used in this study.

ATP concentration was maintained at a constant level in transgenic hearts, almost identical to starting level while significant decrease of ATP has been observed in control hearts. However, this decrease was disproportionately low as compared to deterioration of function or structural changes. Absence of major changes in ATP concentration or Phosphocreatine/Creatine ratio in both transgenic and control hearts indicate good function of energy metabolism. This result is evidence that hyperacute rejection affects predominantly endothelium and vasculature and has no direct effect on myocytes as long as coronary flow is maintained. Use of constant perfusion pressure systems that led to insufficient perfusion could explain why previous studies applying the ex vivo perfusion system demonstrated elevation of cardiac enzymes indicating myocyte damage. The massive increase in UTP concentration that has been observed in control and transgenic hearts at the end of perfusion is intriguing and could be explained by substantially higher uridine concentrations in human blood. However, it is difficult to predict functional consequences of this alteration. One potential effect could be the waste of ATP for uridine phosphorylation reaction but it is unlikely to play a significant role as energy turnover is very high in the heart. Secondly, excess of UTP may disrupt ATP dependent reactions. Furthermore, depending on type of cells that accumulate UTP (endothelium vs. cardiomyocytes), regulatory mechanisms may be affected as it is well established that UTP and ATP are ligands for different types of nucleotide receptors [35].

In conclusion, we have shown that hearts from transgenic pigs produced by SMGT are protected from hyperacute rejection after confrontation with human blood and that those hearts in a human blood environment are relatively metabolically stable and maintain mechanical function above the threshold level for life support. Further application of this method for generation of multi-gene transgenic pigs and in particular its combination with knock-out techniques could be a significant step towards clinical xenotransplantation.


   Notes
 
1 Present address: Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Ave., RN #361, Boston, MA 02215, USA. Back

* This study was supported by the Magdi Yacoub Institute, Grants MiPAF D.M. 427/7303/2002, DM581/7240/96, 564/7240/97, and 404/7240/99, Ministero per la Ricerca Scientifica Grant DD 21.09.99, n462 ric., F.A.R. 2003 Grant University of Milano-Bicocca (to M.L.).

Time for primary review 22 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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