Review

Bioprosthetic heart valves of the future

Manji RA, Ekser B, Menkis AH, Cooper DKC. Bioprosthetic heartvalves of the future. Xenotransplantation 2014: 21: 1–10. © 2014 JohnWiley & Sons A/S.

Abstract: Glutaraldehyde-fixed bioprosthetic heart valves (GBHVs),derived from pigs or cows, undergo structural valve deterioration(SVD) over time, with calcification and eventual failure. It is generallyaccepted that SVD is due to chemical processes between glutaraldehydeand free calcium ions in the blood. Valve companies have made signifi-cant progress in decreasing SVD from calcification through variousvalve chemical treatments. However, there are still groups of patients(e.g., children and young adults) that have accelerated SVD of GBHV.Unfortunately, these patients are not ideal patients for valve replace-ment with mechanical heart valve prostheses as they are at high long-term risk from complications of the mandatory anticoagulation that isrequired. Thus, there is no “ideal” heart valve replacement for childrenand young adults. GBHVs represent a form of xenotransplantation, andthere is increasing evidence that SVD seen in these valves is at least inpart associated with xenograft rejection. We review the evidence thatsuggests that xenograft rejection of GBHVs is occurring, and that calci-fication of the valve may be related to this rejection. Furthermore, wereview recent research into the transplantation of live porcine organs innon-human primates that may be applicable to GBHVs and considerthe potential use of genetically modified pigs as sources of bioprostheticheart valves.

Rizwan A. Manji,1,2 Burcin Ekser,3,4,5

Alan H. Menkis1,2 and David K.C.Cooper31Department of Surgery, University of Manitoba,Winnipeg, MB, Canada, 2Cardiac Sciences Program,Winnipeg Regional Health Authority and St.Boniface Hospital, Winnipeg, MB, Canada, 3ThomasE. Starzl Transplantation Institute, University ofPittsburgh Medical Center, Pittsburgh, PA, USA,4Department of Surgery, Transplantation andAdvanced Technologies, Vascular Surgery and OrganTransplant Unit, University Hospital of Catania,Catania, Italy, 5Department of Surgery, University ofIndianapolis, Indianapolis, IN, USA

Key words: calcification – children/young adults –Gal – genetically modified – glutaraldehyde –heart valves – pigs – xenograft rejection

Abbreviations: Gal, galactose-a1,3-galactose; GBHV,glutaraldehyde-fixed bioprosthetic heart valve;GTKO, a1,3-galactosyltransferase gene-knockout;MHV, mechanical heart valve; NeuGc, N-glycolyl-neuraminic acid; SVD, structural valve deterioration

Address reprint requests to Rizwan A. Manji MD,PhD, MBA, FRCSC, I.H. Asper Clinical ResearchInstitute, St. Boniface Hospital, CR3014 – 369 TacheAvenue, Winnipeg, MB, R2H 2A6 Canada (E-mail:rmanji@sbgh.mb.ca)

Received 19 August 2013;Accepted 25 November 2013

Introduction

Approximately, 20% of all cardiac surgery is forthe treatment of valvular heart disease, and world-wide, more than 250 000 heart valves are replacedeach year [1]. Options for valvular heart surgeryinclude repairing the valve or replacing the valve.Replacement of diseased heart valves has been tak-ing place clinically for >50 years [2]. Replacementof the diseased valve is with either a mechanicalvalve prosthesis (MHV) or a glutaraldehyde-fixedbioprosthetic heart valve (GBHV), usually of por-cine or bovine origin. Worldwide, approximately,55% of replacements are with a MHV and 45%with a GBHV [1].

The decision as to which valve type to implant ina patient depends on a number of variables. When-ever possible, a GBHV is the preferred choicebecause it does not require long-term anticoagu-

lant therapy, with its potential risks, which is man-datory after insertion of a MHV. However,GBHVs tend to have limited durability, especiallyin certain patient populations, such as children andyoung adults [1,3]. Thus, if a GBHV is implantedin these patients, they may require repeat cardiacsurgery within months to a few years. Repeat car-diac surgery is associated with a higher rate ofcomplications and a 2–3-fold higher risk of deaththan the initial operation [4]. Hence, many youngpatients will often receive a MHV if adequate valverepair is not possible.

Assuming there are no complications, for exam-ple, pannus build-up on the valve or infection ofthe valve, MHVs have demonstrated long-termdurability. However, patients with a MHV needlifelong, closely regulated anticoagulation, with itsassociated risks of spontaneous bleeding, thrombo-sis, or thromboembolism, any of which can be

1

Xenotransplantation 2014: 21: 1–10 © 2014 John Wiley & Sons A/S

doi: 10.1111/xen.12080 XENOTRANSPLANTATION

fatal. The cumulative annual risk of bleeding (asso-ciated with therapeutic anticoagulation) is approxi-mately 1% per year [1], with valve thrombosis(from inadequate anticoagulation) being 0.1–5.7%per patient year [1]. Thus, if a patient receives aMHV at age 25, and assuming a 1% bleeding riskand 1% thrombotic risk annually (i.e., a total riskof 2% per year), there is a >99% cumulativechance of either complication by the age of75 years (i.e., within 50 years of the valve surgery).Young patients are also more likely to experiencethese complications as (i) they are more likely to beactive (work/sports) which puts them at greaterrisk of injury and bleeding; (ii) are less reliable intaking medications (including anticoagulants) reg-ularly; (iii) are less likely to adhere to restrictionsto their diet (which is necessary as certain foodscan modify the effectiveness of anticoagulants);and (iv) young women who are menstruating orbecome pregnant may have problems with warfa-rin (currently used for anticoagulation) due tobleeding and the potential of birth defects in thechild. Though newer anticoagulants are now avail-able, which do not have as many restrictions anddo not require monitoring [5], their safety profileand efficacy as they relate to a MHV are unknown,and they are currently not approved for routineuse with a MHV [6].

Glutaraldehyde-fixed bioprosthetic heart valvesare constructed from porcine heart valves or frombovine pericardium that have been glutaraldehyde-treated to help preserve the tissues and decreasetheir immunogenicity [1,2]. A GBHV undergoesstructural valve deterioration (SVD) whereby thevalve thickens and calcifies. This process narrowsthe valve opening, leading to flow-limiting stenosis;in addition, the valve leaflets can tear, which canresult in incompetence (leaking) (Fig. 1). The pro-

cess of calcification is seen in other inflammatoryconditions, such as tuberculosis, and atherosclero-sis, and in these inflammatory conditions, macro-phages and giant cells can be seen on microscopicexamination [7,8]. Furthermore, it is possible thatthe chronic inflammation seen in diseases leads tocalcification through secretion of cytokines bymacrophages, such as osteopontin [9,10]. AsGBHVs are glutaraldehyde-fixed, making themdifficult to destroy and are a xenotransplant, theremay be chronic inflammation in these valves thatleads to calcification.

Structural valve deterioration is age-dependent.In patients >65 years of age, <10% of GBHVs failwithin 10 years, but there is a significant rate offailure within 5 years in patients <35 years of age[1]. It is generally accepted that calcification ofGBHVs occurs from chemical interactions betweenphospholipids, free aldehyde groups and othercomponents of the valve with calcium ions in theblood [11]. The various manufacturers of GBHVshave been able to decrease the rate and extent ofcalcification through several “anti-calcification”chemical processes [11] and have successfullyavoided the development of calcification-relatedSVD in elderly patients. However, children andyoung adults continue to develop early calcifica-tion and failure of GBHVs despite these anti-cal-cification treatments. One intuitive differencebetween non-infant pediatric patients and youngadults compared with the elderly (in whomGBHVs functional longevity is three to four timesthat in young patients) is the immune competenceof the groups [12] and the more rapid metabolism,for example, of calcium, in younger people [1,11].

We here review studies suggesting that there isan immune response to a GBHV, and we highlightlinks between the immune response and calcifica-

Fig. 1. Comparison of an unused glutaraldehyde-fixed bioprosthetic heart valve (GBHV; “off the shelf”) with a GBHV removedfrom a patient for structural valve deterioration. (A) Porcine bioprosthetic heart valve prior to implantation in patient. There is nocalcification and the valve leaflets are thin and clean. (B) Porcine bioprosthetic heart valve removed for structural valve deterioration,showing calcification (C), stenosis of the valve orifice (S), and cusp tears (T).

2

Manji et al.

tion. We also review research into the transplanta-tion of live pig organs into non-human primates asit relates to outcomes after the implantation of aGBHV. Finally, we discuss developments in theproduction of genetically engineered pigs that mayprove to be future sources of GBHVs that offersome protection from the human immune responseand SVD.

Studies suggesting an immune response to a GBHV

Clinical replacement of a diseased heart valve waspioneered in the early 1960s but was accomplishedwith increasing frequency in the 1970s. In the1970s and 1980s, there were reports of inflamma-tory changes (pseudointimal proliferation, fibro-calcific and thrombotic changes, the presence ofactivated mononuclear cells) on histological exami-nation of GBHVs removed from children andyoung adults (2–20 years old) whose GBHVsfailed within 4 years of implantation [2,3,13–17].As early as 1982, Talbert and Wright suggestedthat rejection was occurring in a GBHV (a porcinevalve xenograft) [13].

Electron microscopy and immunohistochemicalstudies by Stein et al. [15] and Wilhelmi et al. [18]added information. Stein et al. [15] used electronmicroscopy to study 33 porcine GBHVs and iden-tified occasional fibroblast-like cells in the valves,platelet deposition in 67%, and leukocyte infil-trates (mostly mononuclear cells) in 82%. The leu-kocytes appeared to have destroyed collagen fibersin the valve and had crystalline material present ontheir surfaces, suggesting they may have been act-ing as a nidus for calcification.

Wilhelmi et al. [18] carried out an immunohisto-chemical analysis of (i) explanted GBHVs, (ii) ex-planted aortic valve allografts (homografts), and(iii) aortic valves of explanted chronically rejectedcardiac allografts, and compared them to normalaortic valves (from unused human donor hearts) ascontrols. The tissues were stained for selectins (e.g.,ELAM-1), integrins (e.g., VLA-1), immunoglobu-lin supergene family members (e.g., ICAM-1 andICAM-2, class I heavy chain proteins), comple-ment adhesion molecules (e.g., CD34, CD44), andvon Willebrand factor. ELAM-1, ICAM-1 andICAM-2, CD34, CD44, and class I heavy chainproteins (all important for inflammatory processes)showed enhanced expression in the allogeneic andxenogeneic valves compared with the control valvesor valves from chronically rejected heart allografts.The explanted allogeneic and xenogeneic valvesshowed strong thrombogenicity, as evidenced bystaining for von Willebrand factor, which waspresent on the surface of endothelial cells.

Animal studies have also suggested that there isa humoral response to a GBHV. Human et al. [19]suggested a role for circulating graft-specific anti-body in causing GBHV calcification. Dahm et al.[20], using enzyme-linked immunosorbent andlymphocyte proliferation assays, provided evidencethat glutaraldehyde-treated bovine pericardialvalves provoked cellular and humoral immuno-logic reactions.

Gabbay et al. [21] and Grabenwoger et al. [22]suggested that the histopathological findings seenrepresented an active immune process and not justa normal healing process. Gabbay et al. [21]implanted glutaraldehyde-fixed pericardial patchxenografts into the left atrial wall in dogs. Thesepatches developed an intense fibrous reaction, cal-cification, and even bone formation on the innersurface in contact with the host’s blood, with mini-mal reaction on the external surface of the patch(not in contact with the host’s blood). Grabenwo-ger et al. [22] examined failed explanted heartvalves from patients who had either had a GBHV(n = 6) or a valve fashioned from autologous peri-cardium treated with glutaraldehyde (autogenic,n = 8). The GBHVs demonstrated large areas ofcalcification and inflammatory infiltration withmacrophages. In contrast, the autologous valvesdid not show any significant inflammatory infil-trate nor any calcification (as evaluated by radio-graphic analysis and microscopic methods) buthad evidence of collagen fiber disruption byplasma proteins and erythrocytes. If the changesseen in this study were strictly related to surgicaltrauma, one would have expected the autologousvalves (which were glutaraldehyde-treated) to showa similar extent of inflammation and calcification.

These combined studies suggest that the inflam-matory/thrombogenic changes seen in GBHVs areindeed an immune response and not simply relatedto surgical healing. The platelet/thrombosis pro-cesses seen on these valves are likely part of theimmune response and not a bystander effect asthere is evidence in the literature suggesting thatthe hemostatic system does have an immuneresponse role as well [23–25]. Figure 2 (left-handside pictorially outlines some of the processes dis-cussed previously).

Studies suggesting a link between the immune response andcalcification

A number of animal studies have suggested a linkbetween inflammation/thrombogenicity/coagula-tion and calcification. Vincentelli et al. [26] pro-vided data that suggested that glutaraldehyde wasnot responsible for graft calcification but that the

3

Future of bioprosthetic heart valves

origin of the tissue (autologous vs. heterologous,i.e., xenogeneic) was the primary factor in thedevelopment of calcification. The study of Humanet al. [19], in which graft-specific antibody wasidentified, supported this conclusion. Figure 2 pic-torially depicts immune response and calcification.A number of studies [18,27,28] have shown thatexperimental models in which the grafted tissue isnot in contact with the blood (non-blood contactmodels, e.g., subcutaneous implantation of thegraft) provide very different results from those inwhich the blood is in contact with the grafted tis-sue, suggesting that, to mimic the human situation,a biological model in which blood is in constantcontact with the grafted tissue is important.

A study in a biological, xenogeneic model, withblood in contact with a glutaraldehyde-fixed valve,in young animals (to mimic clinical GBHV replace-ment in young patients) was conducted by Manjiet al. [27]. In this model, glutaraldehyde-fixed gui-nea pig aortic heart valves with segments ofascending aorta were transplanted into the abdom-inal aorta of young Lewis rats to create a xenoge-neic transplantation model. Some rats were treatedwith corticosteroids to decrease inflammation/rejection. Compared with glutaraldehyde-fixedsyngeneic rat controls, there was significant

destruction of the transplanted valve and aortic tis-sue in the xenogeneic group (suggestive of whatmay occur when a porcine GBHV is implantedinto a child or young adult). This inflammation/rejection was decreased if the rat recipients of theglutaraldehyde-fixed xenogeneic graft were treatedwith steroids (Fig. 3). Compared with the glutaral-dehyde-fixed syngeneic rat controls, there wassignificantly more macrophage and T-cell infiltra-tion into the xenograft tissue, with the pattern ofcellular infiltrate being macrophage infiltration fol-lowed by T-cell infiltration (which is also known tooccur in live-tissue xenotransplantation [29]).Other mononuclear cells were also present in thexenografts, which the authors suggested may havebeen B cells and NK cells, though they did not spe-cifically stain for these [27]. There was evidence ofmicrothrombi on the surface of the valves in thexenogeneic group, but not in the syngeneic group.There was also a rise in rat IgG in the recipients inthe xenogeneic group compared with the syngeneicgroup, and this antibody response was attenuatedif the recipient rats were treated with steroids. Thedifferences between the syngeneic and xenogeneicgroups were statistically significant. This studythus indicated that there were both cellular andhumoral immune responses to the xenogeneic tis-

Fig. 2. Immunological response to GBHV and potential ways a genetically modified BHV may lessen or eliminate the immunologi-cal response. Figure 2 depicts various immunological, coagulative/thrombogenic, and fixative (glutaraldehyde)-related injuries thatcan affect a porcine GBHV, leading to calcification and failure of the graft. Genetic engineering of pigs may reduce or prevent theabove-mentioned injuries. Several groups have reported multiple genetic modifications of the pig, such asGTKO.hCD46.hCD55.hTBM, GTKO.hCD46.CIITA-DN, GTKO.NeuGC-KO, or GTKO.hCD55.hCD39. A20 = tumor necrosisfactor-alpha-induced protein 3; CIITA-DN = MHC class II transactivator-knockdown; CRP = complement regulatory proteins;EC = endothelial cell; ELAM-1 = endothelial-leukocyte adhesion molecule-1; GBHV = glutaraldehyde-fixed bioprosthetic heartvalve; GTKO = a1,3-galactosyltransferase gene-knockout; h = human; HO-1 = heme-oxygenease-1; ICAM = intercellular adhesionmolecule; NeuGc-KO = N-glycolylneuraminic acid gene-knockout; p = porcine TBM = thrombomodulin’; TFPI = tissue factorpathway inhibitor; vWF = von Willebrand factor.

4

Manji et al.

sue, and that this was not simply related to glutar-aldehyde fixation or a foreign body reaction [27].

More importantly, this study also demonstrateda linear relationship between inflammation andcalcification [27]. The extent of calcification corre-lated with the intensity of the cellular infiltrate(especially the macrophage infiltrate). Treating thexenograft group with steroids significantlydecreased the inflammatory infiltrate, the numberof microthrombi, and the extent of calcification.The potential mechanism linking inflammationand calcification may be related to the cytokine,osteopontin, made by macrophages and T cells[30], which is important in the calcification process.Therefore, in a model with relevance to clinicalGBHV implantation, strong evidence was pro-vided linking the inflammatory/immune process tocalcification.

Live xenotransplantation studies and GBHV failure

In 1992, it was reported that the most importantpig antigen to which humans and certain non-human primates have naturally occurring antibod-ies (that initiate hyperacute rejection of pig organs)is galactose-a1,3-galactose (Gal) [31,32]. Anti-Galantibodies are present in mammals that do notexpress Gal (humans, apes and Old World mon-keys), but not in mammals in which Gal isexpressed (e.g., pigs and cows) [33]. It is generally

believed that so-called “natural” or “preformed”antibodies, such as anti-Gal and anti-N-glycolyl-neuraminic acid (NeuGc), develop as a protectiveresponse following colonization of the gastrointes-tinal tract during infancy [34]. All lower ordermammalian species express Gal antigens (andtherefore do not make anti-Gal antibodies). Theproduction of a1,3-galactosyltransferase gene-knockout (GTKO) pigs (which do not express Gal)was a major step forward in overcoming the barri-ers to successful xenotransplantation [35,36]. Thefirst report using GTKO pig hearts in baboons waspublished in 2005 [37]. GTKO pig hearts rarelyundergo hyperacute rejection.

The Gal antigen is expressed on pig endothelialcells, with ~106–107 epitopes per cell [33], and, likeA and B blood group antigens, is probably impor-tant for functions such as cell signaling and cell–-cell interactions [33], though it is clearly notessential. In humans, anti-Gal antibodies are pro-duced by approximately 1% of circulating plasmacells [33]. Anti-Gal IgM comprises 4–8% of totalIgM, and anti-Gal IgG about 1% of total IgG [33].The physiological purpose of anti-Gal antibody isbelieved to be protection against pathogens andmalignant cells, and the removal of senescent orabnormal red blood cells (as these are known toexpress Gal antigens when they become old or dis-eased) [33]. Anti-Gal antibody may also play a rolein human autoimmune disease because in some of

(A) (B)

(C) (D)

Fig. 3. Histopathology following aortic valve and ascending aorta transplantation in rodent models [27]. (A) A syngeneic rat-to-rataortic valve and ascending aorta transplant without any glutaraldehyde fixation (i.e., surgical negative control). (B) A glutaralde-hyde-fixed syngeneic (rat-to-rat) transplant showing mild inflammation. (C) A glutaraldehyde-fixed xenogeneic (guinea pig-to-rat)transplant showing severe inflammation. (D) The reduction in inflammation when the rat recipient of the glutaraldehyde-fixed xeno-geneic (guinea pig-to-rat) transplant is treated with steroids. V = aortic valve; m = media of aortic wall; ad = adventitia of aorticwall. (H&E 9100 for 3A and 9200 for 3B, 3C, 3D).

5

Future of bioprosthetic heart valves

these (e.g., Grave’s disease) Gal is expressed onsome tissues [33].

After overcoming the barrier of hyperacuterejection by addressing Gal, other forms of anti-body-mediated rejection (associated with the pres-ence of anti-non-Gal antibodies), innate immunesystem responses (NK cells and macrophages),cell-mediated responses (T-cell responses), andcoagulation disorders have surfaced as barriers[38,39].

A non-Gal antigen that is almost certain to beimportant in clinical xenotransplantation, and inthe implantation of porcine GBHVs is N-glycolyl-neuraminic acid (NeuGc). This oligosaccharide isexpressed in all mammals with the exception ofhumans [40], and therefore, its importance in theimmune response to a pig graft has been impossi-ble to assess in pig-to-non-human primate organtransplantation models. Nevertheless, in vitrostudies suggest it may play an equivalent role toGal. In the absence of expression of NeuGc ontheir tissues, humans produce anti-NeuGc anti-bodies [41,42], another “natural” antibody [34].Expression of NeuGc on porcine and bovine tis-sues, therefore, may well be an important factor inthe destruction of GBHVs in clinical practice. Veryrecently, pigs that express neither Gal nor NeuGchave been produced [43]. These pigs will almostcertainly play a significant role in future clinical tri-als of pig organ and cell transplantation.

An increasing number of other genetically engi-neered pigs are also becoming established(reviewed in [39,44]) to address the remaining bar-riers to xenotransplantation, including pigs trans-genic for human complement-regulatory,coagulation-regulatory, and/or anti-inflammatoryproteins (Table 1).

As the Gal antigen is so important in live por-cine xenotransplantation (with expression of theantigen on pig tissues and high levels of anti-Galantibody in humans and certain non-human pri-mates), it seemed reasonable to question whetherthe Gal antigen/anti-Gal antibody barrier could beinvolved in the failure of GBHVs. Though GBHVsare not live tissue and thus cannot undergo intrin-sic endothelial cell activation (and its consequentdetrimental sequelae), if prepared from wild-type(i.e., genetically-unmodified) pigs, surface antigens,for example, Gal, even if only expressed on colla-gen and other structural components of the valve,incite an immune response, as do other decellular-ized tissues from these pigs [45].

Several studies have demonstrated the expres-sion of Gal antigens on pig valves and on commer-cially available GBHVs [46], as well as on otherstromal structures [44,47,48]. In fact, Naso et al.

[49] have quantified the expression of Gal epitopeson current GBHVs. A Gal-specific antibodyresponse has also been detected in patients withporcine GBHVs [46]. Gal antigens can be largelyremoved by the enzyme, a-galactosidase [48],reducing injury to the valve by the human immunesystem. Konakci et al. [50] found that immuno-genic Gal epitopes were present on fibrocytes inter-spersed in the connective tissue of porcine valves.Furthermore, patients receiving porcine GBHVsdevelop a significant increase in anti-Gal IgM com-pared to patients with a MHV or those undergoingcoronary artery bypass grafting. These antibodieswere shown to be cytotoxic to Gal-bearing PK-15cells (a porcine kidney cell line). Treating the serawith soluble Gal antigen to adsorb the antibodiesdecreased the cytotoxicity. Jin et al. [51] demon-strated that human monocytes recognize the Galantigen on porcine aortic endothelial cells in cul-ture through the galectin-3 receptor on the mono-cyte. It is not clear if this occurs with GBHVs, butmononuclear cells and macrophages are the pri-mary cellular infiltrate in GBHVs. Thus, the galec-tin-3 (human monocyte)-to-Gal (pig cell)interaction may be important in the inflammatoryresponse seen in GBHVs. Furthermore, there isevidence that the absence of Gal expression on thevascular endothelium decreases the cellularimmune response as well as the humoral response[52].

The link between Gal antigen and calcificationof GBHVs was further suggested by studiesreported by McGregor et al. [53] and Lila et al.[54] who investigated pericardium from GTKOpigs as a potential source for GBHVs. In theseexperiments, GTKO pig pericardium and wild-type pericardium were pre-labeled with humananti-Gal antibody and implanted subcutaneouslyin rodents. The GTKO pig pericardium calcifiedless than did pericardium from wild-type pigs(expressing Gal antigens). The authors concludedthat anti-Gal antibodies interacting with Gal anti-gens on currently used porcine GBHVs may accel-erate the calcification process and suggested thatGTKO pigs may be a source for future GBHVs.The issue with the experiments performed above isthat they were subcutaneous implant models thathave their limitations, as discussed previously (i.e.,do not mimic the human situation with blood flow-ing past the valve).

Several of the other genetic modifications thathave been made in pigs for the purposes of organxenotransplantation (Table 1) may prove benefi-cial to the outcome of a GBHV implanted clini-cally. The NeuGc-knockout pig has already beenmentioned, but pigs expressing complement- and/

6

Manji et al.

or coagulation-regulatory proteins and/or anti-inflammatory genes are also likely to protect fromthe host immune response and thus reduce thespeed and/or extent of calcification. The pig-to-non-human primate model would be the optimalmodel to test GBHVs from these pigs (except forNeuGc-knockout pig grafts, as NeuGc is alsoexpressed in all non-human primates).

Genetically engineered pigs and future GBHVs

There is evidence to suggest that there is rejectionof a GBHV and that pig antigens are involved ininciting the immune response. McGregor et al. [53]and Lila et al. [54] have provided initial evidencethat genetically modified pigs may provideimproved GBHVs in the future. It may soon bepossible to breed pigs that might provide valvesthat are “ideal” for implantation into humans.These valves may not deteriorate structurally orcalcify, or at least these processes may be greatlyslowed. As the valves would be protected, at leastto some extent, from the human immune response,glutaraldehyde treatment may become unnecessary(or less necessary). It is likely that glutaraldehydefixation will inhibit strong function of at least someof these transgenic “protective” proteins, though

the extent of this inhibition is currently unknown.It is likely that, if sufficient protection to the valveis provided by transgenesis, glutaraldehyde fixationmay no longer be required, and “fresh” porcinevalves could be transplanted. However, if geneti-cally engineered “fresh” pig valves were to beimplanted, they would be considered “xenografts”and the breeding, housing, and testing of thesource pigs would be subject to the rigorous regu-lations of the regulatory authorities, which wouldincrease the cost of the valves significantly. Itwould therefore be hoped that this could beavoided by retaining the glutaraldehyde-fixationprocess.

The genetic manipulations that might prove par-ticularly beneficial are those in which human com-plement-regulatory genes (e.g., CD46, CD55, andCD59), coagulation-regulatory genes (e.g., throm-bomodulin, endothelial protein C receptor, tissuefactor pathway inhibitor, CD39), and anti-inflam-matory genes (e.g., heme oxygenase-1, A20) areintroduced (Table 1). In addition, however,manipulations aimed at protecting the graft fromthe human T-cell response may also improve long-term graft survival; in this respect, the introductionof a human mutant MHC class II transactivatorthat results in inhibition of expression of swine leu-kocyte antigen class II, would be valuable, asmight local expression of an immunosuppressivegene, for example, CTLA4-Ig.

The valves could be implanted into children andyoung adults, as well as in older patients, therebyreducing the need for MHVs and their associatedproblems. Figure 2 diagrammatically shows thepresumed mechanisms thought to cause failure ofa GBHV and ways that genetically modified pigsmay resolve the problems. It should be notedthough that if glutaraldehyde fixation is stillneeded, the chemical processes associated with glu-taraldehyde-related injury (left-hand side of Fig. 2)may still occur and will need to be addressed (bythe anti-calcification treatments currently beingundertaken). There may also be new processes thatmay surface as problems following immune-medi-ated injury are addressed.

There are, of course, a potentially large numberof pig antigens against which humans could pro-duce antibodies. Clearly, with present technology,not all of these could be deleted from the pig. How-ever, if the two known key antigens, Gal andNeuGc, are deleted, then there is evidence that theexpression of human complement- and coagula-tion-regulatory transgenes can protect against theinnate immune response to “minor” pig antigens,although exogenous immunosuppressive therapy isrequired to suppress an adaptive immune response.

Table 1. Genetically modified pigs produced for xenotransplantation

researcha

Complement regulation by human complement-regulatory gene expression:

CD46 (membrane cofactor protein)

CD55 (decay-accelerating factor)CD59 (protectin or membrane inhibitor of reactive lysis)

Antigen “masking” or deletion:

Human H-transferase gene expression (expression of blood type O antigen)

Endo-beta-galactosidase C (reduction of Gal antigen expression)

a1,3-galactosyltransferase gene-knockout (GTKO)Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene-

knockout (NeuGc-KO)

Suppression of cellular immune response by gene expression or downregulation

CIITA-DN (MHC class II transactivator knockdown, resulting in swine

leukocyte antigen class II knockdown)

HLA-E/human b2-microglobulin (inhibits human natural killer cell cytotoxicity)Human FAS ligand (CD95L)

Human GnT-III (N-acetylglucosaminyltransferase III) gene

Porcine CTLA4-Ig (Cytotoxic T-Lymphocyte Antigen 4 or CD152)

Human TRAIL (tumor necrosis factor-alpha-related apoptosis-inducing ligand)

Anticoagulation and anti-inflammatory gene expression or deletion

von Willebrand factor (vWF)-deficient (natural mutant)

Human tissue factor pathway inhibitor (TFPI)Human thrombomodulin

Human CD39 (ectonucleoside triphosphate diphosphohydrolase-1)

Anticoagulation, anti-inflammatory, and anti-apoptotic gene expression

Human A20 (tumor necrosis factor-alpha-induced protein 3)

Human heme oxygenase-1 (HO-1)

Prevention of porcine endogenous retrovirus (PERV) activation

PERV siRNA

Combinations of GTKO and expression of transgenes are available (e.g.,

GTKO.hCD46.hCD55).aModified from Ekser B, et al. [39].

7

Future of bioprosthetic heart valves

It would be detrimental to the care of patients withBHVs for exogenous immunosuppressive therapyto be necessary, and so other means of enablinggraft survival would need to be sought, for exam-ple, genetic engineering to introduce the humanMHC class II transactivator mutation. Neverthe-less, if BHV survival could be significantly pro-longed (if not indefinite), this would be hugelybeneficial to young patients in need of valvereplacement.

In addition to the above benefits, physicians areundertaking an increasing number of procedurescarried out percutaneously. Percutaneous tech-niques (that avoid full open surgery) are desirableas the patients experience less pain, and post-oper-ative recovery is much quicker. Recently, percuta-neous transcatheter valve replacements are beingoffered to elderly patients with aortic stenosis feltto be at too high risk of open surgery [55]. The per-cutaneous valves are GBHVs. As experience withthis technique improves, and less invasive proce-dures are demanded by more younger patients,genetically modified bioprosthetic heart valvescould be implanted by these techniques, allowingearlier return to work.

An important hurdle that will need to be over-come will relate to the costs of the use of geneti-cally modified pigs. At present, the raw materialsrequired to fashion a GBHV (i.e., a pig valve orbovine pericardium from wild-type animals) can beobtained from slaughterhouses at manageable, ifnot minimal, cost. In view of the developmentcosts associated with genetic engineering, valvesfrom genetically modified pigs would be signifi-cantly more expensive, but the costs are likely todecrease significantly with expansion of breedingherds. These pigs are originally produced bynuclear transfer/cloning technology but are thenbred naturally, and so expansion of the herds couldbe achieved rapidly and inexpensively. The reducedneed for early GBHV replacement, including hos-pitalization (and, in the case of a MHV, of long-term anticoagulation, with its associated monitor-ing) could offset the increased cost of a geneticallyengineered pig, though this may not have a directfinancial benefit to the manufacturers of eitherform of prosthesis.

Conclusions

This review has provided evidence to suggest thatthere is indeed an immune response to a GBHVand that the rapidity and extent of calcification ofthe valve (which is a very important component ofvalve failure) is correlated with the immuneresponse. Pig antigens that are known to be impor-

tant in graft survival in live pig-to-non-human pri-mate xenotransplantation are almost certainlyplaying a role in the failure of a GBHV (thoughGBHVs are not living tissue). Within the foresee-able future, genetically modified pigs are likely toprovide the “ideal” heart valve for implantationinto humans (especially children and youngadults). If such valves could be fashioned to pro-vide prolonged survival in young patients and inpatients in whom long-term anticoagulation is con-traindicated, there would be a worldwide paradigmshift in valve replacement. Although there is anurgent medical need for such valves—and a hugepotential “market”—the costs of genetically engi-neered pigs have to be considered.

Acknowledgments

Work on xenotransplantation in the Thomas E.Starzl Transplantation Institute of the Universityof Pittsburgh is supported in part by NIH grants #U19 AI090959, # U01 AI068642 and # R21A1074844, and by Sponsored Research Agree-ments between the University of Pittsburgh andRevivicor, Inc., Blacksburg, VA. Burcin EkserMD is a recipient of a NIH NIAID T32 AI 074490Training Grant.

Conflict of interest

The authors have no financial or other conflict ofinterest.

References

1. SIDDIQUI RF, ABRAHAM JR, BUTANY J. Bioprosthetic heartvalves: modes of failure. Histopathology 2009; 55: 135–144.

2. CARPENTIER A, LEMAIGRE G, ROBERT L, CARPENTIER S, DU-

BOST C. Biological factors affecting long-term results ofvalvular heterografts. J Thorac Cardiovasc Surg 1969; 58:467–483.

3. MILLER DC, STINSON EB, OYER PE et al. The durability ofporcine xenograft valves and conduits in children. Circula-tion 1982; 66: I172–I185.

4. RUEL M, CHAN V, BEDARD P et al. Very long-term survivalimplications of heart valve replacement with tissue versusmechanical prostheses in adults <60 years of age. Circula-tion 2007; 116: I294–I300.

5. van de WERF F, BRUECKMANN M, CONNOLLY SJ et al. Acomparison of dabigatran etexilate with warfarin inpatients with mechanical heart valves: the randomized,phase II study to evaluate the safety and pharmacokineticsof oral dabigatran etexilate in patients after heart valvereplacement (RE-ALIGN). Am Heart J 2012; 163: 931–937 e931.

6. STEWART RA, ASTELL H, YOUNG L, WHITE HD. Thrombo-sis on a Mechanical Aortic Valve whilst Anti-coagulatedWith Dabigatran. Heart Lung Circ 2012; 21: 53–55.

7. DOHERTY TM, ASOTRA K, FITZPATRICK LA et al. Calcifica-tion in atherosclerosis: bone biology and chronic inflam-

8

Manji et al.

mation at the arterial crossroads. Proc Natl Acad Sci U SA 2003; 100: 11201–11206.

8. BASARABA RJ, BIELEFELDT-OHMANN H, ESCHELBACH EKet al. Increased expression of host iron-binding proteinsprecedes iron accumulation and calcification of primarylung lesions in experimental tuberculosis in the guinea pig.Tuberculosis (Edinb) 2008; 88: 69–79.

9. CHO HJ, KIM HS. Osteopontin: a multifunctional proteinat the crossroads of inflammation, atherosclerosis, andvascular calcification. Curr Atheroscler Rep 2009; 11: 206–213.

10. SCATENA M, LIAW L, GIACHELLI CM. Osteopontin: a multi-functional molecule regulating chronic inflammation andvascular disease. Arterioscler Thromb Vasc Biol 2007; 27:2302–2309.

11. SIMIONESCU DT. Prevention of calcification in bioprosthet-ic heart valves: challenges and perspectives. Expert OpinBiol Ther 2004; 4: 1971–1985.

12. DIAZ-JAUANEN E, STRICKLAND RG, WILLIAMS RC. Studiesof human lymphocytes in the newborn and the aged. Am JMed 1975; 58: 620–628.

13. TALBERT WM Jr, WRIGHT P. Acute aortic stenosis of a por-cine valve heterograft apparently caused by graft rejection:case report with discussion of immune mediated hostresponse. Tex Heart Inst J 1982; 9: 225–229.

14. PHILLIPS HR, SPRAY TL, LOWE JE, MORRIS KG, WECHSLER

AS. Subvalvular thrombotic obstruction of an aortic por-cine heterograft. Chest 1982; 81: 756–758.

15. STEIN PD, WANG CH, RIDDLE JM, MAGILLIGAN DJ Jr.Leukocytes, platelets, and surface microstructure of spon-taneously degenerated porcine bioprosthetic valves. J CardSurg 1988; 3: 253–261.

16. RIDDLE JM, MAGILLIGAN DJ Jr, STEIN PD. Surface mor-phology of degenerated porcine bioprosthetic valves fourto seven years following implantation. J Thorac Cardio-vasc Surg 1981; 81: 279–287.

17. BUTANY J, ZHOU T, LEONG SW et al. Inflammation andinfection in nine surgically explanted Medtronic Freestylestentless aortic valves. Cardiovasc Pathol 2007; 16: 258–267.

18. WILHELMI MH, MERTSCHING H, WILHELMI M, LEYH R,HAVERICH A. Role of inflammation in allogeneic and xeno-geneic heart valve degeneration: immunohistochemicalevaluation of inflammatory endothelial cell activation. JHeart Valve Dis 2003; 12: 520–526.

19. HUMAN P, ZILLA P. Characterization of the immuneresponse to valve bioprostheses and its role in primary tis-sue failure. Ann Thorac Surg 2001; 71: S385–S388.

20. DAHM M, LYMAN WD, SCHWELL AB, FACTOR SM, FRATER

RW. Immunogenicity of glutaraldehyde-tanned bovinepericardium. J Thorac Cardiovasc Surg 1990; 99: 1082–1090.

21. GABBAY S, BORTOLOTTI U, FACTOR S, SHORE DF, FRATER

RW. Calcification of implanted xenograft pericardium.Influence of site and function. J Thorac Cardiovasc Surg1984; 87: 782–787.

22. GRABENWOGER M, FITZAL F, GROSS C et al. Differentmodes of degeneration in autologous and heterologousheart valve prostheses. J Heart Valve Dis 2000; 9: 104–109; discussion 110-101.

23. HUANG HS, CHANG HH. Platelets in inflammation andimmune modulations: functions beyond hemostasis. ArchImmunol Ther Exp (Warsz) 2012; 60: 443–451.

24. ENGELMANN B, MASSBERG S. Thrombosis as an intravascu-lar effector of innate immunity. Nat Rev Immunol 2012;13: 34–45.

25. TRZECIAK-RYCZEK A, TOKARZ-DEPTULA B, DEPTULA W.Platelets–an important element of the immune system. PolJ Vet Sci 2013; 16: 407–413.

26. VINCENTELLI A, LATREMOUILLE C, ZEGDI R et al. Does glu-taraldehyde induce calcification of bioprosthetic tissues?Ann Thorac Surg 1998; 66: S255–S258.

27. MANJI RA, ZHU LF, NIJJAR NK et al. Glutaraldehyde-fixed bioprosthetic heart valve conduits calcify and failfrom xenograft rejection. Circulation 2006; 114: 318–327.

28. OZAKI S, HERIJGERS P, FLAMENG W. Influence of bloodcontact on the calcification of glutaraldehyde-pretreatedporcine aortic valves. Ann Thorac Cardiovasc Surg 2003;9: 245–252.

29. KIRCHHOF N, SHIBATA S, WIJKSTROM M et al. Reversal ofdiabetes in non-immunosuppressed rhesus macaques byintraportal porcine islet xenografts precedes acute cellularrejection. Xenotransplantation 2004; 11: 396–407.

30. NAU GJ, GUILFOILE P, CHUPP GL et al. A chemoattractantcytokine associated with granulomas in tuberculosis andsilicosis. Proc Natl Acad Sci U S A 1997; 94: 6414–6419.

31. GOOD AH, COOPER DK, MALCOLM AJ et al. Identificationof carbohydrate structures that bind human antiporcineantibodies: implications for discordant xenografting inhumans. Transplant Proc 1992; 24: 559–562.

32. COOPER DK, GOOD AH, KOREN E et al. Identification ofalpha-galactosyl and other carbohydrate epitopes that arebound by human anti-pig antibodies: relevance to discor-dant xenografting in man. Transpl Immunol 1993; 1: 198–205.

33. KOBAYASHI T, COOPER DKC. Anti-Gal, alpha-Gal epi-topes, and xenotransplantation. In: AVILA JL, GALILI U,eds. Anti-Gal: Alpha-1,3 Galactosyltransferase, Alpha-Gal Epitopes, and the Natural Anti-Gal Antibody. Subcel-lular Biochemistry Vol 32. New York: Kluwer Academic/Plenum, 1999: 229–257.

34. GALILI U, MANDRELL RE, HAMADEH RM, SHOHET SB,GRIFFISS JM. Interaction between human natural anti-alpha-galactosyl immunoglobulin G and bacteria of thehuman flora. Infect Immun 1988; 56: 1730–1737.

35. COOPER DK, KOREN E, ORIOL R. Genetically engineeredpigs. Lancet 1993; 342: 682–683.

36. PHELPS CJ, KOIKE C, VAUGHT TD et al. Production ofalpha 1,3-galactosyltransferase-deficient pigs. Science2003; 299: 411–414.

37. KUWAKI K, TSENG YL, DOR FJ et al. Heart transplanta-tion in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat Med2005; 11: 29–31.

38. EZZELARAB M, GARCIA B, AZIMZADEH A et al. The innateimmune response and activation of coagulation inalpha1,3-galactosyltransferase gene-knockout xenograftrecipients. Transplantation 2009; 87: 805–812.

39. EKSER B, EZZELARAB M, HARA H et al. Clinical xenotrans-plantation: the next medical revolution? Lancet 2012; 379:672–683.

40. PADLER-KARAVANI V, VARKI A. Potential impact of thenon-human sialic acid N-glycolylneuraminic acid on trans-plant rejection risk. Xenotransplantation 2011; 18: 1–5.

41. ZHU A. Binding of human natural antibodies to nonalpha-Gal xenoantigens on porcine erythrocytes. Transplanta-tion 2000; 69: 2422–2428.

42. ZHU A, HURST R. Anti-N-glycolylneuraminic acid anti-bodies identified in healthy human serum. Xenotransplan-tation 2002; 9: 376–381.

43. LUTZ AJ, LI P, ESTRADA JL et al. Double knockout pigsdeficient in N-glycolylneuraminic acid and Galactose

9

Future of bioprosthetic heart valves

alpha-1,3-Galactose reduce the humoral barrier to xeno-transplantation. Xenotransplantation 2013; 20: 27–35.

44. COOPER DK, EKSER B, BURLAK C et al. Clinical lung xeno-transplantation–what donor genetic modifications may benecessary? Xenotransplantation 2012; 19: 144–158.

45. DALY KA, STEWART-AKERS AM, HARA H et al. Effect ofthe alphaGal epitope on the response to small intestinalsubmucosa extracellular matrix in a nonhuman primatemodel. Tissue Eng Part A 2009; 15: 3877–3888.

46. PARK CS, PARK SS, CHOI SY et al. Anti alpha-gal immuneresponse following porcine bioprosthesis implantation inchildren. J Heart Valve Dis 2010; 19: 124–130.

47. MCPHERSON TB, LIANG H, RECORD RD, BADYLAK SF.Galalpha(1,3)Gal epitope in porcine small intestinal sub-mucosa. Tissue Eng 2000; 6: 233–239.

48. KASIMIR MT, RIEDER E, SEEBACHER G et al. Presence andelimination of the xenoantigen gal (alpha1, 3) gal in tissue-engineered heart valves. Tissue Eng 2005; 11: 1274–1280.

49. NASO F, GANDAGLIA A, BOTTIO T et al. First quantificationof alpha-Gal epitope in current glutaraldehyde-fixed heartvalve bioprostheses. Xenotransplantation 2013; 20: 252–261.

50. KONAKCI KZ, BOHLE B, BLUMER R et al. Alpha-Gal onbioprostheses: xenograft immune response in cardiac sur-gery. Eur J Clin Invest 2005; 35: 17–23.

51. JIN R, GREENWALD A, PETERSON MD, WADDELL TK.Human monocytes recognize porcine endothelium via theinteraction of galectin 3 and alpha-GAL. J Immunol 2006;177: 1289–1295.

52. WILHITE T, EZZELARAB C, HARA H et al. The effect of Galexpression on pig cells on the human T-cell xenoresponse.Xenotransplantation 2012; 19: 56–63.

53. MCGREGOR CG, CARPENTIER A, LILA N, LOGAN JS, BYRNE

GW. Cardiac xenotransplantation technology providesmaterials for improved bioprosthetic heart valves. J Tho-rac Cardiovasc Surg 2010; 141: 269–275.

54. LILA N, MCGREGOR CG, CARPENTIER S et al. Gal knock-out pig pericardium: new source of material for heart valvebioprostheses. J Heart Lung Transplant 2009; 29: 538–543.

55. LEON MB, SMITH CR, MACK M et al. Transcatheter aor-tic-valve implantation for aortic stenosis in patients whocannot undergo surgery. N Engl J Med 2010; 363: 1597–1607.

10

Manji et al.