Pulmonary xenotransplantation: Rapidly progressing into the unknown

Pulmonary xenotransplantation: Rapidly progressinginto the unknown

Edward Cantu1*, William Parker1, JeffreyL. Platt2 and R. Duane Davis1

1Department of Surgery, Duke University Medical Center,Durham, NC;2Departments of Surgery, Immunology, and Pediatrics,Mayo Clinic, Rochester, MN, USA*Corresponding author: Edward Cantu, [email protected]

As one approach to circumventing the dire shortageof human lungs for transplantation, a handful ofinvestigators have begun to probe the possibility ofpulmonary xenotransplantation. The immunologicand perhaps physiologic barriers encountered bythese investigators are considerable and progress inpulmonary xenotransplantation has lagged behindprogress in cardiac and kidney xenotransplantation.However, during the last few years there have beensubstantial advances in the field of pulmonary xeno-transplantation including, most noticeably, significantprogress in attenuating hyperacute dysfunction. Pro-gress has been made in understanding the barriersimposed by xenoreactive antibodies, complement,coagulation incompatibility and porcine pulmonaryintravascular macrophages. Although our under-standing of the barriers to pulmonary xenotransplant-ation is far from complete and the clinical applicationof pulmonary xenotransplantation is not yet in sight,current progress is fast paced. This progress providesa basis for future work and for a hope that the short-age of human lungs for transplantation will notalways be a matter of life and death.

Received July 17 2003, revised and accepted for pub-lication September 10 2003.

Introduction

In the last four decades following the first clinical lung

transplant (1), advances in immunosuppression and refine-

ments in surgical technique have transformed clinical lung

transplantation into an effective treatment for many

patients with end stage lung disease. Reported cumula-

tive world experience exceeds 14 000 lung transplants

with 73% 1-year and 45% 5-year overall survival (2). The

moderate long-term success achieved, growth of the can-

didate pool and the relatively fixed number of available

donors has resulted in a health crisis where the median

time to transplant has doubled and for every two candidates

who receive an allograft, one dies while still on the

waiting list (3). It has been this trend and the relatively

primitive long-term mechanical replacements available

that have encouraged investigators to evaluate pulmon-

ary xenotransplantation. Such an approach offers a

potential unlimited source of donor organs.

For reasons that have been reviewed elsewhere (4,5),

the pig is currently considered to be the most promising

potential donor for xenotransplantation. In light of this

view, those experiments which model pig-to-human

pulmonary xenotransplantation are considered to be

the most clinically relevant, and are the topic of this

review. Models utilizing pig to primate pulmonary xeno-

transplantation and lung perfusion models using human or

occasionally baboon blood products are discussed. In some

cases, data from other models and from other solid organ

xenotransplants provide information that may provide

insight into pulmonary xenotransplantation, and the implica-

tions of this research for pulmonary xenotransplantation are

discussed.

Hyperactute Pulmonary XenograftDysfunction

The initial experience with pulmonary xenotransplantation

using porcine organs was reported in 1968, prior to the

development of extracorporeal mechanical oxygenators.

Using an ex vivo perfusion apparatus, Bryant and col-

leagues tested pig lungs as potential biologic oxygenators

for humans undergoing cardiac surgical procedures (6).

Unlike the use of lungs from monkeys, the use of swine

lungs was associated with rapid failure of the lung. This

failure was characterized by (a) elevated pulmonary vas-

cular resistance and (b) the rapid development of massive

pulmonary edema evident by gross examination (6). The

presence of these two ‘components’, thrombosis and

edema, has been confirmed by a number of studies

since the initial description in 1968. Histologic examination

of porcine lungs following exposure to human or primate

blood has confirmed the severe pulmonary edema and

demonstrated that the elevated pulmonary vascular resist-

ance is associated with micro- and macrovascular throm-

bosis. Because little evidence existed that immunologic

rejection by the immune system of the recipient was

responsible for pulmonary xenograft failure, the milieu of

thrombosis and edema associated with pulmonary xeno-

transplantation has been termed ‘hyperacute xenograft

dysfunction’. Subsequent studies, described below, have

supported the idea that factors other than the recipient

American Journal of Transplantation 2004; 4 (Suppl. 6): 25–35 Copyright # Blackwell Munksgaard 2004Blackwell Munksgaard

25

immune system play a key role in hyperacute xenograft

dysfunction. Further, the milieu associated with pulmon-

ary xenograft dysfunction is distinct from that associated

with the hyperacute xenograft rejection and the acute

vascular rejection of porcine hearts or kidneys. For these

reasons, the term ‘hyperacute xenograft dysfunction’

rather than the term ‘hyperacute rejection’ or other related

terms is still used to describe the milieu associate with

pulmonary xenograft failure.

In addition to thrombosis and edema, pulmonary xeno-

transplantation has been associated with profound sys-

temic problems. Most noticeably, profound hypotension

occurs in the organ recipient, which requires inotropic

support to maintain the recipient’s hemodynamics in

many experiments. It is likely that this adverse response

is due to a variety of molecules released from the organ,

including C5a and perhaps a number of cytokines, includ-

ing TNFa. In addition, activation of the coagulation cascade

results in a disseminated intravascular coagulopathy (DIC)

that is reversible upon explant of the xenogeneic lung (7).

The coagulopathy is characterized by increased PT, deple-

tion of platelets, fibrinogen, and a dramatic increase in

circulating thrombin-antithrombin complex. Yet another

problem associated with pulmonary xenotransplantation

is a profound depletion of leukocytes from the recipient’s

blood.

A variety of therapies have been successful in eliminating

or delaying one or more of the pathologic process asso-

ciated with pulmonary xenotransplantation (Figures 1–3;

Tables 1 and 2). The results of these experiments and their

implications for the pathogenesis of pulmonary xenograft

dysfunction are described below.

Xenoreactive Antibody and PulmonaryXenotransplantation

There is evidence that xenoreactive antibodies play a role

in hyperacute pulmonary xenograft dysfunction. Pierson

and colleagues, using an ex vivo swine heart-lung model

perfused with human blood, have demonstrated that

increases in pulmonary vascular resistance and pulmonary

edema are associated with immunohistochemical demon-

stration of prominent deposition of IgM and the comple-

ment fragment C4 in the alveolar interstitium, somewhat

less deposition of IgG and patchy deposition of properdin,

C3, and C9. These preliminary studies indicate that xeno-

reactive natural antibody, predominately IgM, binds to the

xenograft and activates complement. Other investigators,

using ex vivo isolated lung perfusion models have repro-

duced these results, finding an association between the

rapid failure of the pulmonary xenografts with deposition

of IgM, IgG, Clq, C3, C4, and C9 (8,9). However, whether

xenoreactive antibody actually remains bound to porcine

endothelium remains in question. Studies performed by

Figure 1: Physiologic response and antibody level in

pulmonary xenotransplantation. (A) Left pulmonary artery

flow; (B) Pulmonary vascular resistance; (C) Percent of

xenoreactive IgM antibody remaining after reperfusion. The key

(top) applies to all three graphs.

Edward Cantu et al.

26 American Journal of Transplantation 2004; 4 (Suppl. 6): 25–35

Parker, Platt and Davis (10) have demonstrated that,

although xenoreactive antibodies can bind to pulmonary

xenografts, many of these antibodies are shed as anti-

body-antigen complexes. A prominent role of these

immune complexes in the activation of complement dur-

ing pulmonary xenotransplantation seems likely, but has

yet to be proven.

Some experiments in which xenoreactive antibody were

depleted from recipients demonstrated improved pulmon-

ary xenograft function in the absence of xenoreactive

antibodies. For example, Pierson and associates demon-

strated that elevated pulmonary vascular resistance could

be prevented with depletion of xenoreactive antibody

utilizing incubation of human plasma with pig splenocytes

Figure 2: Histopathologic abnorm-

alities seen in hyperacute lung

dysfunction. (A) Alveolar edema.

(B) Macrovascular thrombus formation,

pulmonary capillary congestion, and

intra-alveolar congestion. (All stained

with hematoxylin and eosin.)

Figure 3: Summary of in vivo pul-

monary xenotransplantation experi-

ence grouped by time of graft failure

(time on horizontal axis). (pvWF,

porcine von Willebrand Factor; CRPs,

complement regulatory proteins; PIMs,

pulmonary intravascular macrophages;

C5a inhibition, infusion of a monoclonal

anti-C5a antibody.)

Pulmonary xenotransplantation

American Journal of Transplantation 2004; 4 (Suppl. 6): 25–35 27

and peripheral blood cells (11). Macchiarini (12) demons-

trated that prolonged porcine lung survival could be achieved

by first depleting human blood of xenoreactive antibodies

using a porcine lung. Similarly, Lau et al. (13) demonstrated

that depletion of xenoreactive antibodies using an extracor-

poreal lung perfusion resulted in prolonged xenograft survi-

val. However, perfusion of blood through a porcine lung and

perhaps incubation with porcine splenocytes depletes other

components of human and primate blood, including com-

plement, fibrinogen, platelets and leukocytes. Thus, experi-

ments more specifically targeted at xenoreactive antibodies

are required to address the importance of the antibodies in

pulmonary xenotransplantation.

Experiments designed specifically to test the importance of

xenoreactive antibodies in pulmonary xenotransplantation

have not led to the conclusion that xenoreactive antibodies

are critical for pulmonary xenograft dysfunction. For

example, depletion of xenoreactive antibodies by extra cor-

poreal perfusion through a porcine kidney, unlike depletion

using a porcine lung, did not result in prolonged xenograft

survival (13). Similarly, extra corporeal perfusion through

porcine liver or spleen proved unsuccessful in prolong-

ing pulmonary xenograft survival (12). Further, depletion

of anti-Gala1-3Gal antibodies using a column containing

synthetically coupled aGal did not result in prolonged

pulmonary xenograft function. The lack of improved xeno-

graft function in these experiments may be associated

with the release of anaphylatoxins such as C5a, dis-

cussed below. However, hyperacute pulmonary xenograft

dysfunction also occurred in a model using neonatal

primate recipients, which lack anti-Gala1-3Gal antibodies,

Figure 4: Summary of molecular and cellular processes involved in hyperacute lung dysfunction. Antibody, predominantly IgM,

and complement (C) interact with aGal moieties on the endothelial cell surface, resulting in production of terminal complement complexes

(MAC) and in release of anaphlatoxins (C5a, C3a). Pulmonary intravascular macrophages (PIMs) are activated by anaphlatoxins,

xenoreactive antibody plus complement, and/or immune cells, possibly NK cells. Once activated, PIMs release several proinflamatory

molecules and vasoactive eicosanoids, decreasing flow and mediating local and systemic injury. One or more of these interactions gives

rise to endothelial cell activation, which, in turn, results in numerous changes, including loss of heparan sulfate proteoglycan (HSPG) and

of thrombomodulin (TM), increase in tissue factor (TF) expression, and release of high molecular weight von Willebrand Factor (vWF). In

addition, endothelial cell activition results in loss of tight junctions between endothelial cells, resulting in a breakdown of endothelial barrier

function and subsequent edema and cellular infiltration. These changes, coupled with anaphlatoxin-mediated vasoconstriction (pictured at

right), accelerate a procoagulant process that culminates in a thrombosed graft. Also depicted, at right, is the formation of immune

complex between IgM and vWF, which further serves as a nidus for complement activation.

Edward Cantu et al.


indicating that anti-Gala1-3Gal antibodies are not absolutely

required to initiate pulmonary xenograft dysfunction. The

neonatal recipients do, however, contain polyreactive,

xenoreactive antibodies (14,15), suggesting the idea that

antibodies other than anti-Gala1-3Gal antibodies might be

involved in hyperacute pulmonary xenograft dysfunction. On

the other hand, the results may reflect the importance of

factors other than xenoreactive antibodies in pulmonary

xenograft failure. Experiments outlined below utilizing spe-

cific depletion of porcine macrophages from the donor lung

are consistent with this latter interpretation.

Complement in PulmonaryXenotransplantation

Experiments utilizing porcine lungs expressing human

complement regulatory proteins (hCRP) have provided a

wealth of information regarding the role of complement in

pulmonary xenotransplantation. Porcine organs expres-

sing hCRPs, unlike lungs lacking such expression, sub-

stantially attenuate human or primate complement

activation, although perhaps not as efficiently as human

or primate organs (16,17). A number of experiments have

consistently shown improved function of xenografted

porcine lungs expressing hCRP’s vs. those lacking such

expression. For example, Dagget et al. in an ex vivo

perfusion study with human plasma in lungs expressing

DAF and CD59 (Nextran, Princeton, NJ), demonstrated

better function and decreased pulmonary vascular resist-

ance (18) than in non transgenic organs. Similarly, in

Pierson’s ex vivo model, there was a mild prolongation

in xenograft function using swine lungs transgenic for the

hDAF (19). In another series of orthotopic pulmonary

xenotransplants, using swine transgenic for another

CRP, human membrane cofactor protein (MCP, Nextran,

Princeton, NJ), were conducted. As with lungs from

hDAF/CD59 donors, lungs expressing human MCP exhibi-

ted greater blood flow through the xenotransplant, lower

pulmonary vascular resistances, and delayed evidence of

histologic injury as compared to controls expressing no

hCRPs (20).

Although pulmonary xenografts expressing hCRPs fare

better than those lacking hCRPs, they are still subject to

hyperacute pulmonary xenograft dysfunction within hours

(Figure 3). This observation may reflect the idea that

Table 1: Ex vivo pulmonary xenotransplantation experience. Antibody depletion was performed using various strategies prior to

transplant: (1) baboon-to-pig organ perfusion (lung, liver, or spleen); (2) column plasmapheresis (column); or (3) plasma incubation with

aGal antigen (Ag incubation). Transgenic donors were of several types: (1) human decay accelerating factor (hDAF); (2) human CD59; or

(3) combinations thereof. Complement modulation was accomplished either through: (1) heat inactivation; (2) soluble complement

receptor 1 (sCR1); (3) K76-COOH (classical complement activation blockade); (4) FUT-175 (classical and alternative complement activation

blockade). Coagulant modulation was imposed with either heparin or recombinant hirudin (rHirudin)

Donor Recipient n Treatment Survival (h) Reference

Swine Human 7 None 0.2 Azimzadeh et al./2003

10 None 0.5 Azimzadeh et al./2003

4 None 1.9 Wiebe et al./2000

6 None 0.6 Kulick et al./2000

10 None 2.0 Yeatman et al./1999

6 None 2.0 Dagget et al./1997

6 None 1.9 Macchiarini et al./1997

11 None 0.3 Pierson et al./1997

7 None 0.2 Blum et al./1998

6 Ab Depletion(lung perfusion) 5.0 Macchiarini et al./1998

6 Ab Depletion(liver perfusion) 2.5 Macchiarini et al./1998

6 Ab Depletion(spleen perfusion) 1.0 Macchiarini et al./1998

6 Ab Depletion(column) 1.8 Macchiarini et al./1998

3 Ab Depletion(column) 1.5 Pierson et al./1997

12 Transgenic Donor(hDAF) 1.5 Azimzadeh et al./2003

4 Transgenic Donor(hDAF) 4.0 Wiebe et al./2000

5 Transgenic Donor(hCD59) 4.0 Kulick et al./2000

10 Transgenic Donor(hDAF/hCD59) 2.0 Yeatman et al./1999

6 Transgenic Donor(hDAF/hCD59) 2.0 Dagget et al./1997

4 Complement Modulation(sCR1) 0.6 Azimzadeh et al./2003

5 Complement Modulation(heat inactivated) 0.5 Pierson et al./1997

6 Complement Modulation(K76–COOH) 0.6 Blum et al./1998

5 Complement Modulation(FUT�175) 0.8 Blum et al./1998

6 Coagulation Modulation(heparin) <1.0 Schelzig et al./2002

6 Coagulation Modulation(rHirudin) 3.0 Schelzig et al./2002

6 Transgenic Donor(hDAF)/Complement Modulation(heat inactivated) 5.5 Azimzadeh et al./2003

3 Ab Depletion(Ag incubation)/Complement Modulation(heat inactivated) 3.5 Pierson et al./1997



complement regulation by hCRPs is insufficient during

pulmonary xenotransplantation.

Some evidence suggests that pulmonary xenografts may

be uniquely sensitive to complement activation, particu-

larly the anaphylatoxin C5a. Of particular interest are

experiments using cobra venom factor (CVF), a molecule

which depletes complement but produces substantial

amounts of anaphylatoxins, including C5a. Yeatman and

associates, in a pig to baboon single orthotopic lung trans-

plantation model, were only able to show partial protection

from pulmonary xenograft injury in CVF (Quidel, San

Diego, CA) pretreated baboons (21). Using an isolated

working heart–lung perfusion system, Pierson and col-

leagues perfused swine lungs with human blood in order

to determine the mechanisms of pulmonary xenograft dys-

function. Pretreatment of human blood with CVF was

unsuccessful in preventing the development of acute

lung injury (19), consistent with the idea that anaphylatoxin

may injure pulmonary xenografts. Other evidence that

anaphylatoxins may be important in pulmonary xenograft

dysfunction comes from the efficacy of anti-C5a mono-

clonal antibodies (rC5a, Sigma, St.Louis, MO) in prolong-

ing pulmonary xenograft function, although the effect is

short lived (<12 h survival; unpublished data). Although

the lack of efficacy of anticomplement therapy could be

due to an inadequate complement control, other lines

of evidence described below indicate that factors other

than complement play a key role in pulmonary xenograft

dysfunction.

Coagulopathy and Pulmonary XenograftDysfunction

Disseminated intravascular coagulation (DIC) has recently

been proposed as the ‘third’ barrier to xenotransplantation

(22), with xenoreactive antibodies and complement

incompatibility being the first two barriers. DIC is asso-

ciated with the chronic rejection of kidney xenografts

(23,24), developing over a period of weeks in these xeno-

grafts. DIC is also associated with acute processes follow-

ing bone marrow xenotransplantation (25), developing

over a period of days following bone marrow xenotrans-

plantation. In contrast, DIC is observed within minutes to

hours of pulmonary xenograft reperfusion (7).

Several incompatibilities between the human and porcine

coagulation systems have been identified as potential

underlying causes of the coagulopathy associated with

pulmonary xenotransplantation. One potential incom-

patibility is that porcine von Willebrand Factor (vWF) inter-

acts in an aberrant fashion with human platelets. Von

Willebrand Factor is a protein stored by platelets and

endothelial cells that is released upon activation of those

cells (26–28). In normal individuals, platelet activation and

adhesion occurs when vWF binds to GPIb on platelets

only if the platelets are subjected to shear stress (29–

34). In contrast, porcine von Willebrand Factor (pvWF)

binds human (or primate) GPIb on quiescent platelets,

leading to platelet aggregation even in the absence of

shear stress (35,36). Thus, aberrant interactions between

Table 2: In vivo pulmonary xenotransplantation experience. Antibody depletion was performed using various strategies prior to

transplant: (1) baboon-to-pig lung perfusion (lung perfusion); (2) column plasmapheresis (column); or (3) aGal conjugated to polyethelene

glycol (aGal-PEG). Transgenic donors were of several types: (1) human membrane cofactor protein (hMCP); (2) human decay accelerating

factor (hDAF); (3) human CD59 (hCD59); or (4) combinations thereof. Macrophage depletion strategies utilized an encapsulated heavy

metal chelator (clodronate), which selectively destroys macrophages by induction of apoptosis

Donor Recipient n Treatment Survival (h) Reference

Swine Baboon 3 None <3 Yeatman et al./1999

3 None <3 Dagget et al./1998

2 None <1 Norin et al./1996

6 None <72 Kaplon et al./1995

3 None <3 Lau et al./2003

3 CVF <1 Yeatman et al./1999

4 Ab Depletion(lung perfusion) 11 Dagget et al./1998

4 Transgenic Donor (hDAF/CD59) 3 Yeatman et al./1999

4 Transgenic Donor(hDAF/CD59) 3 Lau et al./2000

3 Transgenic Donor(hMCP) <2 Gaca et al./2002

4 Transgenic Donor(hMCP) 12 Gonzalez-Stawinski et al./2002

3 Transgenic Donor(hCD59) <12 Norin et al./1996

2 vWFd <3 Lau et al./2003

2 Transgenic Donor(hDAF/CD59)/Ab Depletion(column) <1 Lau et al./2000

1 Transgenic Donor(hMCP)/Ab Depletion(lung perfusion) <1 Gaca et al./2002

4 Transgenic Donor(hMCP)/Ab Depletion(column) 4 Gonzalez-Stawinski et al./2002

5 Transgenic Donor(hMCP)/Ab Depletion(aGAL–PEG) 4 Gonzalez-Stawinski et al./2002

3 Transgenic Donor(hMCP)/antiGP1b <3 Gaca et al./2002

1 Transgenic Donor(hMCP)/Ab Depletion(column)/antiC5a 9 Gaca et al./2002

5 Transgenic Donor(hMCP)/Ab Depletion(aGAL–PEG)/PIM depletion(Clodronate) 22.6 Unpublished

3 vWFd Donor/Ab Depletion(aGAL–PEG)/PIM depletion(Clodronate) 67 Unpublished

Edward Cantu et al.


pvWF and human or primate GPIb could lead to wide-

spread activation of the coagulation system, resulting in

DIC.

The interaction between pvWF and human GPIb may be

particularly important in pulmonary xenotransplantation, as

evidence suggests that pulmonary xenografts shed more

pvWF than either heart or kidney xenografts (10). As a

direct test of this idea, the interaction between pvWF

and primate GPIb in the DIC associated with pulmonary

xenotransplantation was blocked in a series of swine-

to-baboon pulmonary xenotransplants using a monoclonal

antibody to GPIb (monoclonal Lp-J3) (37). Treatment with

the anti-GPIb antibody resulted in a loss of platelets,

through splenic sequestration. Treatment with anti-GPIb

antibody in splenectomized baboons (normal number of

platelets) and in baboons with intact spleens (elimination

of platelets) prevented the decreases in fibrinogen and

increases in D-dimers observed in control xenotransplants,

indicating that activation of platelets by porcine vWF plays

a significant role in the DIC associated with pulmonary

xenograft dysfunction.

On the other hand, elevations in the PT and generation of

thrombin were not prevented by anti-GPIb therapy (37),

suggesting that activation of the coagulation system

occurs by means other than or in addition to the interac-

tion of pvWF with platelets. Indeed, there are at least two

other molecular incompatibilities between porcine and

human proteins that might cause or at least contribute to

coagulopathy. Porcine TFPI, which is present in lung tis-

sues (38), and is a modulator of thrombosis in the lung

(39), does not inhibit human factor Xa (40,41). Further,

porcine TM does not activate the anticoagulant human

(or primate) protein C (42). Thrombomodulin may be pre-

sent in lung tissues to a greater extent than in other

tissues (43), suggesting that problems associated with

the ineffectiveness of porcine TM may be especially pro-

nounced in pulmonary xenotransplantation compared to

heart and kidney xenotransplantation.

Role of Pulmonary IntravascularMacrophages in Pulmonary XenograftDysfunction

Swine lungs differ from primate lungs because, in addition

to the resident pulmonary alveolar macrophages, there are

pulmonary intravascular macrophages (PIMs) that com-

prise more than 16% of microvascular surface area (44).

Importantly, these pulmonary intravascular macrophages

(PIMs) are largely absent in humans and primates. PIMs

are a resident population of macrophages, morphologically

resembling Kupffer cells, which are tightly adherent to the

endothelium by cell junctions and are not readily dis-

placed. Whether they can be induced in humans or

primates remains controversial.

The physiologic function of these cells is to filter blood of

any foreign material or bacteria passing through the lung, a

process, which normally takes place in the liver or spleen

in species without large populations of PIMs (45–48). In

addition to their scavenging function, PIMs, produce

arachidonate metabolites including thromboxane, cytokines

including IL-1, IL-2, and TNF-a, and procoagulant factors,

including tissue factor and PAI-1 (48–50). In acute lung

injury and some xenotransplants models evidence sug-

gests PIMs may contribute significantly to the rapid devel-

opment of pulmonary hypertension and edema (45,47,51).

Using a technique to deplete macrophages using lipo-

somal clodronate (Roche, Mannheim, Germany) developed

by Van Rooijen (52,53), Staub was able to reliably deplete

>90% of the PIM population in sheep (54). In the PIM

depleted sheep, there was no pulmonary vasoconstrictive

response to particle infusion. In PIM depleted vs. non-

depleted sheep, LPS infusion resulted in complete abroga-

tion of increases in PVR and >90% attenuation of the

capillary leak (54,55).

Pierson and colleagues using lungs from swine treated with

liposomal clodronate for their heterologous perfusion experi-

ments, found decreased production of thromboxane and

preservation of a PVR comparable to the homologous con-

trol perfusion. In addition, PIM depletion decreased platelet

sequestration, C3a levels, TNF-a release, and prolonged

pulmonary function (50). More recently, Gaca and associ-

ates using an in vivo pig to primate orthotopic lung transplant

model with PIM depletion have been able to abrogate the

consumptive coagulopathy seen in controls and extend graft

function to 24 h (Figure 3, data unpublished).

Hypothetical Barriers

There are a number of hypothetical barriers to pulmonary

xenotransplantation. Perhaps most concerning is a

hypothetical problem common to all xenografts – xoono-

sis. Considerable attention has been placed on the pos-

sibility that infectivity of transplanted pig organs may place

the community as a whole at risk (5). Although cross-

species transmission of diseases has and continues to

be a health problem, the transmission of disease in pig-

to-human transplantation is a hypothetical problem. Many

consider that following strict housing and breeding guide-

lines would make the risk of transferring known swine

infections less likely (56). Further, selective breeding or

gene targeting strategies to further reduce the risk of

known infections are likely possible (57). Thus, given our

current level of understanding, community risk of known

infections is minimal (5); however, it is also recognized

that a cavalier attitude is inappropriate, and that we should

proceed with caution (58–62).

Another hypothetical problem is that physiologic incompat-

ibility may pose a barrier to pulmonary xenotransplantation.



Although porcine organs have been able to support

primates without the assistance of a native lung or other

means of oxygenation (63), there are some known physio-

logic differences between porcine and human lungs. For

example, porcine lungs contain less substantial collateral

channels and more musculature in the arterial walls than

do human lungs (64). This should make pulmonary hyper-

tension more of a concern in xenografts than in allografts.

On the other hand, because collateral resistance is much

greater than airway resistance, the collateral pathways do

not apparently play a ‘significant role’ under normal physio-

logic circumstances, even in humans (64). Thus, it remains

unknown what effect a decrease in collateral pathways

might have on a xenograft recipient.

Another hypothetical concern regarding pulmonary xeno-

transplantation is that, being an immune organ, the lung

may itself need to be substantially immunosuppressed to

facilitate engraftment, and that such immunosuppression

may lead to infection of the organ. The fact that elimination

of PIMs from the organ dramatically improves xenograft

function substantiates the idea that immunosuppression

of the organ may be required. This hypothetical problem

will likely materialize or be dismissed as the duration of

xenograft survival in clinically relevant models is extended.

Yet another hypothetical barrier to pulmonary xenotrans-

plantation is one that has already been encountered in

heart and kidney xenotransplantation. Specifically, the

recipient may mount a T-cell dependent response to the

graft, resulting in the production of high affinity IgG

specific for the porcine tissue. It is hoped that, if indeed

this barrier is encountered, therapies currently showing

promise in heart and kidney xenotransplantation will prove

equally successful in pulmonary xenotransplantation.

Future Directions

The hurdles to the application of clinical pulmonary xeno-

transplantation are substantial, and even though signifi-

cant advances have been made, it seems apparent that

work toward that application is only beginning. Preliminary

results from our group indicate that depletion of PIMs

combined with attenuation of xenoreactive antibodies

and elimination of porcine vWF (vWF-deficient swine

bred at the University of North Carolina, Chapel Hill, NC)

can prolong pulmonary xenograft survival up to five days,

even in the absence of complement inhibition. Although

the etiology of graft failure in the absence of PIMs appears

similar to the more rapid graft failure observed in the

presence of the PIMs, the mechanisms underlying this

failure remain unknown. The failure may be due, at least

in part, to PIMs which were either not depleted or which

are regenerated. In addition, factors such as complement

activation and problems with coagulation regulation prob-

ably play a role in the failure of PIM-depleted lungs just as

in PIM-sufficient lungs. However, the presence of other

barriers has not been ruled out and future studies will

necessarily address this issue. In addition, the ability of

lungs depleted of PIMs to withstand infection will need to

be addressed.

Inevitably, experiments aimed at achieving tolerance in pul-

monary xenotransplantation will take place. However, suc-

cess in this area is not a short-term goal. Work toward

tolerance to porcine kidney xenografts is progressing but

is facing serious hurdles (65–67). It is anticipated that

work in this area with porcine lungs may prove even

more challenging, given that the lung is, like the gut, an

immune organ.

Future studies will necessarily address therapies to over-

come the problems with coagulation regulation in pulmon-

ary xenotransplantation. Although vWF deficiency has

proven effective in our model, the ultimate goal is to

block aberrant interactions between porcine vWF and

gp1b without eliminating the normal function of vWF.

Achieving this goal may provide us with insight into other

problems with coagulation regulation that need to be

addressed. Importantly, if early events unrelated to coagu-

lation initiate the associated DIC, the complex nature of

the DIC may be less important from a clinical perspective

if the early events can be blocked. Thus, future studies in

pulmonary xenotransplantation should be aimed not only

at understanding the factors that contribute directly to

DIC, but also to factors that potentially initiate the process.

The future of xenotransplantation is inexorably tied to the

development of genetic engineering in the pig. Cloning of

pigs has recently been achieved and provides new and

promising avenues of investigation. Healthy galactosyl a1–3galactosyl transferase deficient swine have been born

earlier this year (68), and this technology may now be

applied to other molecules (69). Gene transfer technolo-

gies in pigs have recently advanced, allowing for the ability

to either knock out or knock in genes of interest quicker,

with greater efficiency, and with less expense (70–76).

Our increasing ability to create genetically modified pigs,

coupled with our rapidly improving understanding of the

immunology surrounding pulmonary xenograft dysfunc-

tion, fuel the hope that pulmonary xenotransplantation

will one day be a viable option for patients with end

stage pulmonary disease.

Conclusion

Substantial progress has been made in the last 10 years

regarding the role of xenoreactive antibodies, comple-

ment, coagulopathy and, more recently, pulmonary intra-

vascular macrophages in the pathologic milieu associated

with pulmonary xenotransplantation. Although this review

has treated each of these factors separately, the interplay

between them is certainly an integral part of the patholo-

gical process (Figure 4). For example, porcine vWF shed

Edward Cantu et al.


from pulmonary xenografts may play a key role not only in

the coagulopathy and DIC associated with pulmonary

xenotransplantation, but, when complexed with xenoreac-

tive antibodies, may be a significant factor in the produc-

tion of C5a. This anaphylatoxin, in turn, may interact with

C5a receptors present on macrophages (77) thus leading

to macrophage stimulation and subsequent increases in

the proinflammatory and procoagulant milieu (78).

Despite this progress, the limited survival of pulmonary

xenografts that is currently observed in large animal

models means that the clinical application of pulmonary

xenotransplantation is not yet in sight, and, although none

can foresee what lies around the next corner, much addi-

tional work is required.

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Pulmonary xenotransplantation: Rapidly progressing into the unknown