The Endothelial Glycocalyx in Pig-to-Baboon Cardiac Xenotransplantation—First Insights

Abstract

Cardiac xenotransplantation has seen remarkable success in recent years and is emerging as the most promising alternative to human cardiac allotransplantation. Despite these achievements, acute vascular rejection still presents a challenge for long-term xenograft acceptance and new insights into innate and adaptive immune responses as well as detailed characterizations of signaling pathways are necessary. In allotransplantation, endothelial cells and their sugar-rich surface—the endothelial glycocalyx—are known to influence organ rejection. In xenotransplantation, however, only in vitro data exist on the role of the endothelial glycocalyx so far. Thus, in the current study, we analyzed the changes of the endothelial glycocalyx components hyaluronan, heparan sulfate and syndecan-1 after pig-to-baboon cardiac xenotransplantations in the perioperative (n = 4) and postoperative (n = 5) periods. These analyses provide first insights into changes of the endothelial glycocalyx after pig-to-baboon cardiac xenotransplantation and show that damage to the endothelial glycocalyx seems to be comparable or even less pronounced than in similar human settings when current strategies of cardiac xenotransplantation are applied. At the same time, data from the experiments where current strategies, like non-ischemic preservation, growth inhibition or porcine cytomegalovirus (a porcine roseolovirus (PCMV/PRV)) elimination could not be applied indicate that damage of the endothelial glycocalyx also plays an important role in cardiac xenotransplantation.

Full text

Citation: Bender, M.; Abicht, J.-M.;
Reichart, B.; Leuschen, M.; Wall, F.;
Radan, J.; Neumann, E.; Mokelke, M.;
Buttgereit, I.; Michel, S.; et al. The
Endothelial Glycocalyx in Pig-to-
Baboon Cardiac Xenotransplantation—
First Insights. Biomedicines 2024,12,
1336. https://doi.org/10.3390/
biomedicines12061336
Academic Editor: Anand
Prakash Singh
Received: 7 May 2024
Revised: 10 June 2024
Accepted: 14 June 2024
Published: 16 June 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
biomedicines
Article
The Endothelial Glycocalyx in Pig-to-Baboon Cardiac
Xenotransplantation—First Insights
Martin Bender 1, * , Jan-Michael Abicht 1, Bruno Reichart 2, Maria Leuschen 2, Felicia Wall 2, Julia Radan 2,
Elisabeth Neumann
2
, Maren Mokelke
2
, Ines Buttgereit
1
, Sebastian Michel
3,4
, Reinhard Ellgass
3
, Katja Gieseke
1
,
Stig Steen 5, Audrius Paskevicius 5, Joachim Denner 6, Antonia W. Godehardt 7, Ralf R. Tönjes 7,
Christian Hagl 3,4, David Ayares 8, Eckhard Wolf 9,10,11 , Michael Schmoeckel 3, Paolo Brenner 3,
Martin B. Müller 1, † and Matthias Längin 1, †
1Department of Anaesthesiology, University Hospital, LMU Munich, 81377 Munich, Germany
2Transregional Collaborative Research Center 127, Walter Brendel Centre of Experimental Medicine,
LMU Munich, 81377 Munich, Germany
3Department of Cardiac Surgery, University Hospital, LMU Munich, 81377 Munich, Germany
4Munich Heart Alliance, German Center for Cardiovascular Research (DZHK), 81377 Munich, Germany
5
Department of Cardiothoracic Surgery, Lund University and Skåne University Hospital, 221 85 Lund, Sweden
6Institute of Virology, Free University Berlin, 14163 Berlin, Germany
7Division of Haematology, Cell and Gene Therapy, Paul-Ehrlich-Institut, 63225 Langen, Germany
8Revivicor, Blacksburg, VA 24060, USA
9Institute of Molecular Animal Breeding and Biotechnology, Gene Center, and Department of Veterinary
Sciences, LMU Munich, 81377 Munich, Germany
10 Center for Innovative Medical Models (CiMM), LMU Munich, 81377 Munich, Germany
11 Interfaculty Center for Endocrine and Cardiovascular Disease Network Modelling and Clinical
Transfer (ICONLMU), LMU Munich, 81377 Munich, Germany
*Correspondence: martin.bender@med.uni-muenchen.de; Tel.: +49-89-2180-76507
†These authors contributed equally to this work.
Abstract: Cardiac xenotransplantation has seen remarkable success in recent years and is emerging
as the most promising alternative to human cardiac allotransplantation. Despite these achievements,
acute vascular rejection still presents a challenge for long-term xenograft acceptance and new in-
sights into innate and adaptive immune responses as well as detailed characterizations of signaling
pathways are necessary. In allotransplantation, endothelial cells and their sugar-rich surface—the
endothelial glycocalyx—are known to influence organ rejection. In xenotransplantation, however,
only
in vitro
data exist on the role of the endothelial glycocalyx so far. Thus, in the current study,
we analyzed the changes of the endothelial glycocalyx components hyaluronan, heparan sulfate
and syndecan-1 after pig-to-baboon cardiac xenotransplantations in the perioperative (n = 4) and
postoperative (n = 5) periods. These analyses provide first insights into changes of the endothelial
glycocalyx after pig-to-baboon cardiac xenotransplantation and show that damage to the endothelial
glycocalyx seems to be comparable or even less pronounced than in similar human settings when
current strategies of cardiac xenotransplantation are applied. At the same time, data from the ex-
periments where current strategies, like non-ischemic preservation, growth inhibition or porcine
cytomegalovirus (a porcine roseolovirus (PCMV/PRV)) elimination could not be applied indicate
that damage of the endothelial glycocalyx also plays an important role in cardiac xenotransplantation.
Keywords: heart; xenotransplantation; endothelial glycocalyx; endothelial activation; organ preservation;
orthotopic heart transplantation
1. Introduction
Cardiac xenotransplantation has seen remarkable success in recent years and is emerg-
ing as the most promising alternative to human cardiac allotransplantation [
1
–
3
]. This
Biomedicines 2024,12, 1336. https://doi.org/10.3390/biomedicines12061336 https://www.mdpi.com/journal/biomedicines

Biomedicines 2024,12, 1336 2 of 15
success was made possible by essential achievements and important findings in differ-
ent
in vivo
and
in vitro
models [
4
–
6
]: the development of genetically modified donor
pigs lacking surface sugar antigens as well as expressing different human genes [
1
,
7
],
immunosuppression based on co-stimulation blockade of the CD40/CD40 ligand (CD40L)
pathway [
8
], continuous cold non-ischemic heart preservation [
9
,
10
], the relevance of
growth
control [4,11–13]
, the absence of porcine cytomegalovirus (a porcine roseolovirus
(PCMV/PRV)) [
14
,
15
] and other pathogens in the donor animals [
1
,
16
] and the relevant role
of inflammatory responses and coagulation disorders following
xenotransplantation [17–20]
.
Using these current strategies, consistent survival for up to nine months was achieved
in life-supporting pig-to-baboon experiments [
4
–
6
] and the first pig-to-human cardiac
xenotransplantations were performed as individual medical treatments in 2022 [
21
,
22
] and
2023 [23].
Despite these outstanding achievements, acute vascular rejection still presents a chal-
lenge for long-term xenograft acceptance [
24
] and new insights into innate and adaptive
immune responses as well as detailed characterizations of signaling pathways are necessary.
In our pig-to-baboon xenotransplantation experience, attempts to reverse acute vascular re-
jection using high-dose steroid therapy have proven uniformly unsuccessful [
1
]. Therefore,
we suggest that attention should be directed to safely preventing acute vascular rejection
as well as developing and testing potential treatment options in pre-clinical models [1].
Endothelial dysfunction and persistent endothelial inflammation are known as hall-
marks of acute vascular rejection in allogenic transplantation [
25
]. Endothelial health is
closely linked to an intact endothelial sugar-rich surface layer known as the endothelial
glycocalyx [
26
], while endothelial dysfunction is not only accompanied but also reinforced
by the degradation of the endothelial glycocalyx [
27
]. It is mainly composed of core
proteoglycans from the syndecan and glypican families, carrying highly sulfated, linear
glycosaminoglycan attachments, including heparan sulfate and chondroitin sulfate, as
well as non-sulfated receptor-bound hyaluronan [
28
,
29
]. Endothelial glycocalyx integrity
plays a major role in preventing acute vascular rejection by limiting inflammation and
maintaining endothelial homeostasis [
30
]. In human liver [
31
], lung [
32
], and kidney [
33
]
allotransplantation, damage to the endothelial glycocalyx, indicated by elevated plasma
concentrations of glycocalyx breakdown products, is correlated with reduced organ sur-
vival and early graft rejection. In xenotransplantation, however, only
in vitro
data exist on
the role of the endothelial glycocalyx so far [
34
–
36
], indicating that glycocalyx shedding is
linked to complement activation and xenograft rejection [
35
,
36
], while protection against
complement activation contributes to maintaining an intact glycocalyx layer on endothelial
cells [34].
To broaden these initial
in vitro
findings and to gain further insights into the role of
the endothelial glycocalyx in cardiac xenotransplantation, we retrospectively analyzed
the changes in the plasma concentrations of the major endothelial glycocalyx components
hyaluronan, heparan sulfate and syndecan-1 as surrogate biomarkers for glycocalyx in-
tegrity following pig-to-baboon cardiac xenotransplantation.
2. Materials and Methods
2.1. Animals and Study Groups
Hearts from five genetically modified piglets were transplanted into male baboons.
The piglets (German Landrace/Large White; blood group 0) were homozygous for alpha1,3-
galactosyltransferase knockout (GGTA1-KO) and hemizygous transgenic for human CD46
(hCD46) and human thrombomodulin (hTBM) (Revivicor, Blacksburg, VA, USA and Insti-
tute of Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich,
Germany). Five baboons (Papio anubis and Papio hamadryas; blood group B; German Primate
Centre (DPZ), Göttingen, Germany) served as recipients. Expression of hCD46 and hTBM
was verified post mortem by immunohistochemistry. Two animals were tested positive for
PCMV/PRV, as published elsewhere [5,14].

Biomedicines 2024,12, 1336 3 of 15
Four baboons, #17186, #17290, #17494 and #17492, were analyzed in the perioperative
and postoperative period after pig-to-baboon cardiac xenotransplantation. These studies
were divided into two groups in the postoperative analyses: those that were deliberately
terminated after 90 postoperative days (#17186 and #17290, group I) and animals that were
tested positive for PCMV/PRV (#17494 and #17492, group II). As probes were available
for the postoperative period (but perioperative probes were no longer available), a fifth
baboon, #16755, was analyzed in this period. In this animal, the xenograft was ischemically
preserved and no growth-inhibiting drugs were administered (see below), as described in
detail elsewhere [4].
The study was approved by the Government of Upper Bavaria. All animals were cared
for and treated in accordance with the Guide for the Care and Use of Laboratory Animals
(German Legislation for the Welfare of Laboratory Animals and US National Institutes
of Health).
2.2. Anesthesia, Surgical Procedure and Heart Preservation
After sedation, induction of anesthesia and endotracheal intubation of the animals [
37
],
surgery was conducted as published in detail elsewhere [4].
In brief, after median sternotomy of the donor animal, the aorta was cross-clamped.
In one animal, #16755, the heart was perfused with a single dose of 20 mL/kg crystalloid
Belzer’s UW cardioplegic solution (Preservation Solutions, Elkhorn, WI, USA) at 4
◦
C. The
appendices of the right and left atrium were opened for decompression. The heart was
then excised, submersed in cardioplegic solution and stored on ice. In four animals, #17186,
#17290, #17494 and #17492, antegrade non-ischemic preservation commenced immediately
after cross-clamping of the aorta; continuous perfusion with 8
◦
C oxygenated, hyperoncotic
solution containing albumin, hormones, nutrients and erythrocytes [
9
,
10
] was provided
by an extracorporeal heart preservation system (University of Lund, Sweden) consisting
of a pressure- and flow-controlled roller pump, an O
2
/CO
2
exchanger, a leukocyte filter
and a cooler/heater unit. During storage, the heart was preserved the same way and the
perfusion pressure kept at 20 mmHg.
After median sternotomy in the baboon recipient, extracorporeal circulation was
installed and started. Explantation of the recipient’s native heart and xenotransplantation
followed the techniques of Lower and Shumway [
38
]. In the four animals with non-ischemic
preservation, the donor heart was intermittently perfused for 2 min every 15 min during
implantation, as described in detail elsewhere [10].
2.3. Immunosuppression, Anti-Inflammatory and Additive Therapy
Immunosuppressive therapy was based on a CD40/CD40L co-stimulation blockade [
8
].
For induction therapy, all animals received B cell depleting anti-CD20 ab (Mabthera; Roche
Pharma, Basel, Switzerland), anti-thymocyte globulin (ATG, thymoglobulin; Sanofi, Paris,
France) and a mouse/rhesus chimeric anti-CD40 IgG4 monoclonal antibody (anti-CD40
Mab; 50 mg/kg body weight (bw); mouse/rhesus chimeric IgG4 clone 2C10R4, NIH
Non-human Primate Reagent Resource; Mass Biologicals, Boston, MA, USA; courtesy of
K. Reimann).
Immunosuppression was maintained with mycophenolate mofetil (CellCept; Roche
Pharma, Basel, Switzerland), methylprednisolone (urbasone soluble; Sanofi, Paris, France)
and anti-CD40 Mab (50 mg/kg bw once weekly).
All animals received anti-inflammatory therapy including an C1 esterase inhibitor
(Berinert; CSL Behring, King of Prussia, PA, USA), an interleukin 6 (IL-6) receptor antagonist
(RoActemra; Roche Pharma, Basel, Switzerland), a TNF
α
inhibitor (Enbrel; Pfizer, New
York, NY, USA) and an IL-1 receptor antagonist (Kineret; Swedish Orphan Biovitrum, Solna,
Sweden) [4,19].
The additive medication consisted of acetylsalicylic acid (Aspirin; Bayer, Leverkusen,
Germany) and unfractionated heparin (Heparin-Natrium-25000-ratiopharm
®
; Ratiopharm,
Ulm, Germany). Furthermore, ganciclovir (Cymevene, Roche Pharma, Basel, Switzer-

Biomedicines 2024,12, 1336 4 of 15
land), cefuroxime (Cefuroxim; Hikma Pharmaceuticals, London, UK) and epoetin beta
(NeoRecormon 5000; Roche Pharma, Basel, Switzerland) were also administered [4].
Four animals, #17186, #17290, #17494 and #17492, received a therapeutic regime to slow
xenograft overgrowth, which was described in detail elsewhere [
4
,
5
]. Methylprednisolone
was tapered down quickly and additional antihypertensive drugs (enalapril (Enahexal;
Hexal, Holzkirchen, Germany) and metoprolol tartrate (Beloc; AstraZeneca, Cambridge,
UK)) as well as the mTOR inhibitor temsirolimus (Torisel; Pfizer, New York, NY, USA)
were added.
2.4. Blood Sampling and Lactate Measurements
Blood samples were taken from baboon recipients prior to xenotransplantation, regu-
larly during each experiment and before euthanasia. Lactate measurements were performed
with Siemens RAPIDLab
®
1200 Systems (Siemens, Munich, Germany). The measurements
before the beginning of surgical procedures on the day of xenotransplantation were defined
as baseline (Pre XTx).
2.5. Measurement of Endothelial Glycocalyx Components
The concentrations of the shedded endothelial glycocalyx components in the plasma
samples were analyzed using the following enzyme-linked immunosorbent assays: Human
CD138 ELISA Kit (Diaclone SAS, Besançon, France) detects natural and recombinant human
Syndecan-1 protein without cross reactivity with other human soluble molecules; the HS
ELISA Kit (Wuhan Fine Biotech Co., Ltd., Wuhan, China) specifically recognizes heparan
sulfate with no obvious cross reaction with other analogues, according to the manufacturer;
the Hyaluronan Enzyme-Linked Immunosorbent Assay Kit (Echelon Biosciences Inc., Salt
Lake City, UT, USA) detects HA molecules that are as small as 6.4 kDa [
39
]. ELISAs
were performed according to the manufacturer’s protocol. The intra- and inter-assay
variability for each ELISA kit is indicated by the coefficient of variation, as provided by the
manufacturer: HS: 5.2%; 5.3%, HA: <20%; <10%, and Syndecan-1: 6.2%; 10.2%.
2.6. Statistics
Data collection and analyses were performed with Excel 2019 (Microsoft, Redmond,
WA, USA) and GraphPad Prism 9.0 (GraphPad Software Inc., Boston, MA, USA). Data are
presented either as single measurements or as group means
±
SD if not indicated otherwise.
3. Results
We present analyses of the three endothelial glycocalyx components—the two gly-
cosaminoglycans, hyaluronan and heparan sulfate, as well as the proteoglycan
syndecan-1—in
the perioperative (n = 4) and postoperative period (n = 5) after pig-to-baboon orthotopic
cardiac xenotransplantation experiments. Other data from these experiments, e.g., pre-
and postoperative immunologic parameters, causes of death and myocardial histological
findings have not been subject to this retrospective data analysis and have been published
in detail elsewhere [4,5,14]. Some of these data are summarized in Table 1.
Table 1. Overview of the study group. F, female; M, male; PCMV/PRV, porcine cytomegalovirus/
porcine roseolovirus.
Experiment
DPZ-ID
Donor Recipient
Group Survival Growth
Inhibition Preservation Causes for Euthanasia
Sex Weight Sex Weight
1 #16755 M 15.8 kg M16.0 kg -30 days No Ischemic Heart and liver
failure [4]
2 #17186 F 19.3 kg M21.5 kg I90 days Yes Non-ischemic Study endpoint [4,5]
3 #17290 F 12.7 kg M13.7 kg I90 days Yes Non-ischemic Study endpoint [4,5]
4 #17494 M 11.6 kg M16.0 kg II 15 days Yes Non-ischemic Multiorgan failure
(PCMV/PRV) [5,14]
5 #17492 F 24.0 kg M26.0 kg II 27 days Yes Non-ischemic Multiorgan failure
(PCMV/PRV) [5,14]

Biomedicines 2024,12, 1336 5 of 15
Two experiments, #17186 and #17290 (group I), were deliberately terminated when
the predetermined period of 90 postoperative days (set by the regulatory authorities) was
reached, with the animals in excellent clinical condition [
4
,
5
]. Two animals, #17494 and
#17492 (group II), were tested positive for PCMV/PRV [
14
] and experiments were termi-
nated after 15 and 27 days, respectively, because they presented with signs of multiorgan
failure [
5
]. In contrast to the other animals, baboon #16755 received a heart that was is-
chemically preserved and no growth inhibitory drugs were administered. This animal
developed progressive diastolic left ventricular failure because of myocardial hypertrophy
and associated terminal liver disease [4].
3.1. Baseline Values of the Circulating Plasma Endothelial Glycocalyx Components
Baseline hyaluronan levels (Pre XTx) were 140.197
±
24.430 ng/mL, baseline heparan
sulfate levels were 11,534.683
±
4480.468 ng/mL and baseline syndecan-1 levels were
23.117 ±5.228 ng/mL (Figure 1a,c,e).
Biomedicines 2024, 12, x FOR PEER REVIEW 5 of 16
histological findings have not been subject to this retrospective data analysis and have been
published in detail elsewhere [4,5,14]. Some of these data are summarized in Table 1.
Table 1. Overview of the study group. F, female; M, male; PCMV/PRV, porcine
cytomegalovirus/porcine roseolovirus.
Experiment DPZ-ID Dono
r
Recipient
Group Survival Growth
Inhibition Preservation Causes for Euthanasia
Sex Weight Sex Weight
1 #16755 M 15.8 kg M 16.0 kg - 30 days No Ischemic Heart and liver failure
[4]
2 #17186 F 19.3 kg M 21.5 kg I 90 days Yes Non-ischemic Study endpoint [4,5]
3 #17290 F 12.7 kg M 13.7 kg I 90 days Yes Non-ischemic Study endpoint [4,5]
4 #17494 M 11.6 kg M 16.0 kg II 15 days Yes Non-ischemic Multiorgan failure
(PCMV/PRV) [5,14]
5 #17492 F 24.0 kg M 26.0 kg II 27 days Yes Non-ischemic Multiorgan failure
(PCMV/PRV) [5,14]
Two experiments, #17186 and #17290 (group I), were deliberately terminated when
the predetermined period of 90 postoperative days (set by the regulatory authorities) was
reached, with the animals in excellent clinical condition [4,5]. Two animals, #17494 and
#17492 (group II), were tested positive for PCMV/PRV [14] and experiments were
terminated after 15 and 27 days, respectively, because they presented with signs of
multiorgan failure [5]. In contrast to the other animals, baboon #16755 received a heart
that was ischemically preserved and no growth inhibitory drugs were administered. This
animal developed progressive diastolic left ventricular failure because of myocardial
hypertrophy and associated terminal liver disease [4].
3.1. Baseline Values of the Circulating Plasma Endothelial Glycocalyx Components
Baseline hyaluronan levels (Pre XTx) were 140.197 ± 24.430 ng/mL, baseline heparan
sulfate levels were 11,534.683 ± 4480.468 ng/mL and baseline syndecan-1 levels were
23.117 ± 5.228 ng/mL (Figure 1a,c,e).
(a) (b)
(c) (d)
Biomedicines 2024, 12, x FOR PEER REVIEW 6 of 16
(e) (f)
Figure 1. Perioperative changes of hyaluronan (a,b), heparan sulfate (c,d) and syndecan-1 (e,f) in
absolute values (ng/mL) and as fold increases compared to the preoperative values (left and right,
respectively). (b,d,f) Mean values ± SD (n = 4). CPB, cardiopulmonary bypass; POD1, first
postoperative day; XTx, xenotransplantation.
3.2. Perioperative Changes of the Endothelial Glycocalyx Components
In the perioperative period, levels of hyaluronan, heparan sulfate and syndecan-1
showed a consistent course in all four baboons (Figure 1a,c,e).
Hyaluronan levels showed no relevant change 1 h after cardiopulmonary bypass
(CPB) was stopped as compared to the baseline levels. When measured 6 h after
termination of CPB, hyaluronan levels increased 2.5-fold as compared to the baseline
levels and stayed at these increased levels during the first postoperative day (Figure 1a,b).
Heparan sulfate and syndecan-1 decreased in the perioperative period (Figure 1c–f).
Compared to their baseline levels, heparan sulfate presented with a decrease around of
0.5-fold at 1 h and 6 h after CPB and during the first postoperative day (Figure 1c,d). The
decrease in syndecan-1 levels was less pronounced, compared to heparan sulfate (Figure
1e,f).
3.3. Perioperative Lactate Changes and Correlation with Endothelial Glycocalyx Components
Similar to the endothelial glycocalyx components, perioperative lactate levels also
showed a consistent course in all four baboons (Figure 2a,b). Starting with absolute values
around of 1.0 mmol/L at the beginning of surgery, lactate levels increased about 1.5-fold
1 h and about 2-fold 6 h after CPB was stopped. At the first postoperative day, the levels
returned to baseline or even lower. There was no significant correlation between the
perioperative lactate levels and the levels of hyaluronan (Figure 2c), heparan sulfate
(Figure 2d) or syndecan-1 (Figure 2e).
(a) (b)
Figure 1. Perioperative changes of hyaluronan (a,b), heparan sulfate (c,d) and syndecan-1 (e,f) in
absolute values (ng/mL) and as fold increases compared to the preoperative values (left and right,
respectively). (b,d,f) Mean values
±
SD (n = 4). CPB, cardiopulmonary bypass; POD1, first postoper-
ative day; XTx, xenotransplantation.

Biomedicines 2024,12, 1336 6 of 15
3.2. Perioperative Changes of the Endothelial Glycocalyx Components
In the perioperative period, levels of hyaluronan, heparan sulfate and syndecan-1
showed a consistent course in all four baboons (Figure 1a,c,e).
Hyaluronan levels showed no relevant change 1 h after cardiopulmonary bypass (CPB)
was stopped as compared to the baseline levels. When measured 6 h after termination of
CPB, hyaluronan levels increased 2.5-fold as compared to the baseline levels and stayed at
these increased levels during the first postoperative day (Figure 1a,b).
Heparan sulfate and syndecan-1 decreased in the perioperative period (Figure 1c–f).
Compared to their baseline levels, heparan sulfate presented with a decrease around of
0.5-fold at 1 h and 6 h after CPB and during the first postoperative day (Figure 1c,d).
The decrease in syndecan-1 levels was less pronounced, compared to heparan sulfate
(Figure 1e,f).
3.3. Perioperative Lactate Changes and Correlation with Endothelial Glycocalyx Components
Similar to the endothelial glycocalyx components, perioperative lactate levels also
showed a consistent course in all four baboons (Figure 2a,b). Starting with absolute
values around of 1.0 mmol/L at the beginning of surgery, lactate levels increased about
1.5-fold
1 h and about 2-fold 6 h after CPB was stopped. At the first postoperative day, the
levels returned to baseline or even lower. There was no significant correlation between
the perioperative lactate levels and the levels of hyaluronan (Figure 2c), heparan sulfate
(Figure 2d) or syndecan-1 (Figure 2e).
Biomedicines 2024, 12, x FOR PEER REVIEW 6 of 16
(e) (f)
Figure 1. Perioperative changes of hyaluronan (a,b), heparan sulfate (c,d) and syndecan-1 (e,f) in
absolute values (ng/mL) and as fold increases compared to the preoperative values (left and right,
respectively). (b,d,f) Mean values ± SD (n = 4). CPB, cardiopulmonary bypass; POD1, first
postoperative day; XTx, xenotransplantation.
3.2. Perioperative Changes of the Endothelial Glycocalyx Components
In the perioperative period, levels of hyaluronan, heparan sulfate and syndecan-1
showed a consistent course in all four baboons (Figure 1a,c,e).
Hyaluronan levels showed no relevant change 1 h after cardiopulmonary bypass
(CPB) was stopped as compared to the baseline levels. When measured 6 h after
termination of CPB, hyaluronan levels increased 2.5-fold as compared to the baseline
levels and stayed at these increased levels during the first postoperative day (Figure 1a,b).
Heparan sulfate and syndecan-1 decreased in the perioperative period (Figure 1c–f).
Compared to their baseline levels, heparan sulfate presented with a decrease around of
0.5-fold at 1 h and 6 h after CPB and during the first postoperative day (Figure 1c,d). The
decrease in syndecan-1 levels was less pronounced, compared to heparan sulfate (Figure
1e,f).
3.3. Perioperative Lactate Changes and Correlation with Endothelial Glycocalyx Components
Similar to the endothelial glycocalyx components, perioperative lactate levels also
showed a consistent course in all four baboons (Figure 2a,b). Starting with absolute values
around of 1.0 mmol/L at the beginning of surgery, lactate levels increased about 1.5-fold
1 h and about 2-fold 6 h after CPB was stopped. At the first postoperative day, the levels
returned to baseline or even lower. There was no significant correlation between the
perioperative lactate levels and the levels of hyaluronan (Figure 2c), heparan sulfate
(Figure 2d) or syndecan-1 (Figure 2e).
(a) (b)
Biomedicines 2024, 12, x FOR PEER REVIEW 7 of 16
(c) (d)
(e)
Figure 2. Perioperative courses of serum lactate in absolute values (ng/mL) and as fold increases
compared to start of surgery (a,b). Correlation between perioperative lactate levels and changes of
hyaluronan (c), heparan sulfate (d) and syndecan-1 (e). (b), mean values ± SD (n = 4). CPB,
cardiopulmonary bypass; POD1, first postoperative day; XTx, xenotransplantation.
3.4. Postoperative Changes of the Endothelial Glycocalyx Components
In contrast to the consistent courses in the perioperative period, postoperative
changes of the glycocalyx components were different in the animals whichwere
deliberately terminated after 90 postoperative days with the baboons in excellent clinical
condition [4] (group I) compared to the baboons with PCMV/PRV infections [5] (group II)
(Figure 3).
POD 1
POD 6/7
POD 12-14
POD 20-22
POD 26-28
POD 42
POD 56
POD 70
POD 82-84
POD 90
0
2,000
4,000
6,000
8,000
10,000
12,000
Postoperative time
Group I
Group II
#16755
(a)
Figure 2. Perioperative courses of serum lactate in absolute values (ng/mL) and as fold increases
compared to start of surgery (a,b). Correlation between perioperative lactate levels and changes
of hyaluronan (c), heparan sulfate (d) and syndecan-1 (e). (b), mean values
±
SD (n = 4). CPB,
cardiopulmonary bypass; POD1, first postoperative day; XTx, xenotransplantation.

Biomedicines 2024,12, 1336 7 of 15
3.4. Postoperative Changes of the Endothelial Glycocalyx Components
In contrast to the consistent courses in the perioperative period, postoperative changes
of the glycocalyx components were different in the animals whichwere deliberately ter-
minated after 90 postoperative days with the baboons in excellent clinical condition [
4
]
(group I) compared to the baboons with PCMV/PRV infections [5] (group II) (Figure 3).
Biomedicines 2024, 12, x FOR PEER REVIEW 7 of 16
(c) (d)
(e)
Figure 2. Perioperative courses of serum lactate in absolute values (ng/mL) and as fold increases
compared to start of surgery (a,b). Correlation between perioperative lactate levels and changes of
hyaluronan (c), heparan sulfate (d) and syndecan-1 (e). (b), mean values ± SD (n = 4). CPB,
cardiopulmonary bypass; POD1, first postoperative day; XTx, xenotransplantation.
3.4. Postoperative Changes of the Endothelial Glycocalyx Components
In contrast to the consistent courses in the perioperative period, postoperative
changes of the glycocalyx components were different in the animals whichwere
deliberately terminated after 90 postoperative days with the baboons in excellent clinical
condition [4] (group I) compared to the baboons with PCMV/PRV infections [5] (group II)
(Figure 3).
POD 1
POD 6/7
POD 12-14
POD 20-22
POD 26-28
POD 42
POD 56
POD 70
POD 82-84
POD 90
0
2,000
4,000
6,000
8,000
10,000
12,000
Postoperative time
Group I
Group II
#16755
(a)
Biomedicines 2024, 12, x FOR PEER REVIEW 8 of 16
(b)
(c)
Figure 3. Postoperative courses of hyaluronan (a), heparan sulfate (b) and syndecan-1 (c) plasma
concentrations. Group I, experiments deliberately terminated after 90 postoperative days with the
baboons in excellent clinical condition, mean values ± SEM (n = 2); Group II, baboons with
PCMV/PRV infections, mean values ± SEM (n = 2). PCMV/PRV, porcine
cytomegalovirus/roseolovirus; POD, postoperative day.
In the group I animals, all glycocalyx components showed a stable postoperative
course without relevant increases or decreases until the end of the experiments after 90
postoperative days (Figure 3a–c).
In contrast to group I, in group II animals, hyaluronan levels showed a slight increase
in the first postoperative week followed by a sharp increase in the second postoperative
week to values over 10,000 ng/mL, representing about a 100-fold increase compared to the
baseline levels (Figure 3a). In group II, heparan sulfate was stable in the first postoperative
week and then decreased to levels of around 3000 ng/mL until the end of the experiments
(Figure 3b). Syndecan-1 levels presented with a sharp increase in the first postoperative
week to levels of around 30 ng/mL and remained at these levels until the end of the group
II experiments (Figure 3c).
In baboon #16755, hyaluronan levels were stable until the end of the second
postoperative week. Afterwards, hyaluronan increased to values of around 5000 ng/mL
until the end of the experiment (Figure 3a). Heparan sulfate ranges were lower as
compared to group I and group II, with values of around 3000 ng/mL in the first
postoperative days. Within three postoperative weeks, heparan sulfate increased to 6000
ng/mL, followed by a sharp decrease until the end of the experiment (Figure 3b).
Syndecan-1 was stable in the first postoperative week and then increased to levels of
around 20 ng/mL until the end of the experiment (Figure 3c).
Figure 3. Postoperative courses of hyaluronan (a), heparan sulfate (b) and syndecan-1 (c) plasma
concentrations. Group I, experiments deliberately terminated after 90 postoperative days with the ba-
boons in excellent clinical condition, mean values
±
SEM (n = 2); Group II, baboons with PCMV/PRV
infections, mean values
±
SEM (n = 2). PCMV/PRV, porcine cytomegalovirus/roseolovirus; POD,
postoperative day.
In the group I animals, all glycocalyx components showed a stable postoperative
course without relevant increases or decreases until the end of the experiments after
90 postoperative days (Figure 3a–c).

Biomedicines 2024,12, 1336 8 of 15
In contrast to group I, in group II animals, hyaluronan levels showed a slight increase
in the first postoperative week followed by a sharp increase in the second postoperative
week to values over 10,000 ng/mL, representing about a 100-fold increase compared to the
baseline levels (Figure 3a). In group II, heparan sulfate was stable in the first postoperative
week and then decreased to levels of around 3000 ng/mL until the end of the experiments
(Figure 3b). Syndecan-1 levels presented with a sharp increase in the first postoperative
week to levels of around 30 ng/mL and remained at these levels until the end of the group II
experiments (Figure 3c).
In baboon #16755, hyaluronan levels were stable until the end of the second postop-
erative week. Afterwards, hyaluronan increased to values of around 5000 ng/mL until
the end of the experiment (Figure 3a). Heparan sulfate ranges were lower as compared to
group I and group II, with values of around 3000 ng/mL in the first postoperative days.
Within three postoperative weeks, heparan sulfate increased to 6000 ng/mL, followed by a
sharp decrease until the end of the experiment (Figure 3b). Syndecan-1 was stable in the
first postoperative week and then increased to levels of around 20 ng/mL until the end of
the experiment (Figure 3c).
4. Discussion
4.1. Baseline Values of the Circulating Plasma Endothelial Glycocalyx Components
The hyaluronan and syndecan-1 baseline values in the current study group are com-
parable to the human setting. In healthy humans, circulating median concentrations of
126.0 ng/mL were described for hyaluronan [
40
,
41
] and 29.5 ng/mL for syndecan-1 [
41
,
42
],
when measured with the same ELISA used in the current study. However, the heparan
sulfate baseline level of 11,534.7 ng/mL in the current study is slightly higher than the
average values described in three human studies that reported mean concentrations of 4800,
5590 and 7000 ng/mL respectively [
43
–
45
]. Importantly, in these studies, healthy human
individuals showed a great heterogeneity in plasma heparan sulfate concentrations [
43
–
46
].
The higher baseline heparan sulfate levels could be partly due to the strikingly higher base-
line value of 18,164.2 ng/mL in animal #17492. Furthermore, it should also be considered
that a different heparan sulfate ELISA was used in our study, in comparison to the human
study mentioned above.
4.2. Perioperative Changes of the Endothelial Glycocalyx Components
Several human studies showed an increase of the circulating glycocalyx components
hyaluronan, heparan sulfate and syndecan-1 in adult and infant patients undergoing car-
diac [
40
,
41
,
47
–
50
] and major vascular surgery [
44
]. For example, in a human cardiac surgery
study, hyaluronan ranges increased about 5-fold when CPB was stopped (a timepoint not
available in the current study) and were still about 2-fold higher at 1 h after termination
of CPB [
40
]. In another study in humans undergoing cardiac surgery, hyaluronan lev-
els increased about 1.5-fold at 1 h and about 1.3-fold at 6 h after CPB was stopped and
returned to their preoperative levels on the second postoperative day [
48
]. As the time
points assessed were not identical, these human data cannot be directly compared with our
analyses. However, it appears as if the perioperative hyaluronan changes in the current
group occurred with a time delay compared to the human data—while they showed no
relevant change 1 h after CPB in the current study group, hyaluronan levels increased about
2.5-fold at 6 h after CPB was stopped and on the first postoperative day.
In contrast to the human data mentioned above [
40
,
44
,
47
,
48
], where heparan sulfate
levels also increased significantly in the perioperative period, heparan sulfate decreased
about 0.5-fold in the current study group at 1 h and 6 h after CPB and on the first postoper-
ative day. This was also seen in another human study during early reperfusion in patients
undergoing cardiac surgery [
50
]. In this study, heparan sulfate decreased by 14% during
the first minute after aortic declamping and thereafter remained below the pre-reperfusion
level [
50
]. A decrease of heparan sulfate was also seen in human liver allotransplanta-
tion after restoration of the splanchnic and lower body circulation [
31
]. Heparan sulfate

Biomedicines 2024,12, 1336 9 of 15
and other glycosaminoglycans adhere rapidly to the damaged glycocalyx [
51
–
53
], which
could explain a decrease of circulating heparan sulfate levels. Furthermore, in human
hemorrhage, endogenous glycocalyx preservation coincided with a decrease in circulating
heparan sulfate [
54
]. We therefore assume that the heparan sulfate decrease in the current
study could have been caused by rapid endogenous restoration of the glycocalyx. As the
recipient baboons all received protamine after CPB, the decrease of heparan sulfate could
also be explained by removal of protamine-bound heparan sulfate from the circulation [
55
].
Although, to our knowledge, there are no human data in this regard, we assume
that the perioperative decrease of syndecan-1 in the current study was caused by similar
mechanisms as the decrease of heparan sulfate.
Summarizing our perioperative findings, we suggest that there was less glycoca-
lyx shedding in the current study group than in comparable human cardiac surgery
studies. In the human setting, the shedding of the endothelial glycocalyx is mainly at-
tributed to ischemia/reperfusion injury [
56
–
58
], inflammation induced by TNF
α
[
59
],
the release of atrial natriuretic peptides [
40
,
60
] and is furthermore seen as general phe-
nomenon after CPB [
48
,
61
]. In all four animals of the perioperative study group, cold
non-ischemic heart preservation with continuous perfusion was applied [
10
]. As this
prevents ischemia/reperfusion injury [
10
], the non-ischemic preservation could explain
the reduced glycocalyx shedding in the current study group. In addition to avoiding
ischemia/reperfusion injury, the preservation solution containing albumin [
4
,
9
,
10
] could
be an important factor in this regard. Addition of albumin to the preservation solution
improved endothelial integrity and heart performance in guinea pigs, which was partly
explained by the protective effects of albumin on the endothelial glycocalyx [
62
]. Further-
more, the anesthetic agent sevoflurane, which has proved protective to the endothelial
glycocalyx [63–65], was used in all animals.
However, with only four animals, the present analysis can only provide initial insights
and indications regarding glycocalyx shedding in cardiac xenotransplantation. Further
studies with larger numbers of cases and possibly further analyses are needed to answer
these questions in more detail.
4.3. Perioperative Lactate Changes and Correlation with Endothelial Glycocalyx Components
The changes in perioperative lactate levels in the current study group were comparable
to existing human data [
48
]. In humans, during CPB in cardiac surgery, lactate levels as a
parameter of the microcirculation also showed about a 2-fold increase in the perioperative
period, which correlated significantly with perioperative syndecan-1 changes [
48
]. This
correlation could be explained by microcirculatory perfusion disturbances caused by the
perturbation of the endothelial glycocalyx [
56
,
66
,
67
]. There was no correlation between
perioperative lactate changes and changes of hyaluronan, heparan sulfate or syndecan-1 in
the current study group, although lactate changes were comparable to the human study
mentioned above [
48
]. This could be explained by less perioperative shedding of the
endothelial glycocalyx in the current study group (see above). However, it is also possible
that the study group was too small to produce such a correlation.
4.4. Postoperative Changes of the Endothelial Glycocalyx Components
There was no relevant change in the levels of hyaluronan, heparan sulfate or syndecan-
1 in the two experiments of group I. At best, there was a slight increase in hyaluronan
towards the end of the experiments. Both animals showed an unremarkable clinical course
without any signs of rejection and were deliberately terminated after 90 days [
4
,
5
]. We
therefore interpret the current data as a sign that there is no relevant damage to the endothe-
lial glycocalyx after pig-to-baboon cardiac xenotransplantation using current strategies
of CD40/CD40L based immunosuppression [
8
], organ preservation [
9
,
10
] and growth
inhibition [
4
]. Furthermore, both baboons in group I received an IL-6 receptor antagonist, a
TNF
α
inhibitor and a C1 esterase inhibitor as part of the anti-inflammatory regimen [
4
,
19
].
In humans, the IL-6 receptor antagonist tocilizumab improved the endothelial glycocalyx

Biomedicines 2024,12, 1336 10 of 15
in rheumatoid arthritis patients [
68
] and application of a TNF
α
inhibitor protected against
endotoxin-induced endothelial glycocalyx perturbation [
69
]. Regarding potential beneficial
effects of the C1 esterase inhibitor,
in vitro
data from genetically modified porcine endothe-
lial cells suggest that protection against complement activation contributes to maintaining
an intact endothelial glycocalyx [
34
]. The slight increase in hyaluronan levels in group I
animals could be explained by recurring (bacterial) infections [
70
] treated with antibiotics.
The animals in group II both were tested positive for PCMV/PRV [
14
,
15
] and had to
be euthanized because of multiorgan failure after 15 and 27 postoperative days, respec-
tively [
5
]. In these animals, hyaluronan and syndecan-1 levels showed a marked increase,
whereas heparan sulfate levels decreased until the end of the experiments. We assume
that these changes were caused, at least in part, by infection with PCMV/PRV. To our
knowledge, there are so far no data on the interaction of PCMV/PRV with the endothelial
glycocalyx. However, there are data on other virus infections leading to damage of the
endothelial glycocalyx in humans [
71
–
75
], for example COVID-19 [
71
], H1N1 influenza [
72
],
hanta [
75
] and dengue virus [
73
,
74
]. For example, in H1N1 influenza infections, elevated
hyaluronan levels were associated with an increase in mortality rate [
76
] and increased
plasma syndecan-1 levels were an independent risk factor for mortality [
77
]. In the case
of PCMV/PRV, high levels of tissue plasminogen activator and inhibitor 1 complexes in
baboons transplanted with a PCMV/PRV-positive pig heart indicate a complete loss of
the pro-fibrinolytic properties of the endothelial cells [
14
]. These findings and the fact that
PCMV/PRV does not infect human cells [
78
] suggest that PCMV/PRV may directly interact
with endothelial cells. Furthermore, an increased level of IL-6 was found in PCMV/PRV-
positive animals #17494 and #17492 [
14
]. These increased IL-6 levels may have contributed
to the glycocalyx damage seen after infection by the influenza virus H1N1 [
72
]. Proinflam-
matory cytokines, such as IL-6, are known to activate enzymes named sheddases, which
induce glycocalyx degradation [79,80].
The fifth animal in the postoperative study group, #16755, had to be euthanized
because of heart and liver failure due to myocardial overgrowth after 30 postoperative
days [
4
]. Comparable to group II, hyaluronan and syndecan-1 increased and heparan sulfate
decreased, thus indicating damage to the endothelial glycocalyx. As the donor heart was
ischemically preserved in this animal, this could have been caused by ischemia/reperfusion
injury [
56
–
58
]. However, we assume that the increase should already have been noticeable
on the first postoperative day, if ischemia/reperfusion injury was the cause. Since this
was not the case, we assume that the changes in the endothelial glycocalyx were caused
by the complications of myocardial overgrowth of the xenograft [
4
,
11
]. Currently, there
are no data on glycocalyx degradation and cardiac xenograft overgrowth. However, in
human patients with heart failure of other entities damage to the glycocalyx has been
described [81,82].
4.5. Limitations
The number of experimental animals in the current retrospective analysis is limited
and there is no “classical” control group. Therefore, the available data do for example
not allow for any further statistical analyses. However, considering the principles of the
3 Rs [
83
], the high value of donor pigs and recipient baboons, and the complexity of the
experiments, we believe these data are worth publishing and adequate for gaining first
insights into the endothelial glycocalyx in pig-to-baboon cardiac xenotransplantation.
We investigated glycocalyx injury based on the measurement of circulating glycocalyx
components in the plasma. Since it is still a matter of debate whether these soluble glyco-
calyx components correlate adequately with glycocalyx structure and function [
84
], the
plasma concentrations of hyaluronan, heparan sulfate and syndecan-1 presented here can
only be interpreted as surrogate biomarkers for glycocalyx integrity.
Further, ideally, prospective studies with more experiments and possibly additional
analyses should be performed to obtain a deeper understanding of the endothelial glycoca-
lyx in cardiac xenotransplantation.

Biomedicines 2024,12, 1336 11 of 15
5. Conclusions
The current analysis provides first insights into changes of the endothelial glycocalyx
after pig-to-baboon cardiac xenotransplantation. Using current strategies of cardiac xeno-
transplantation, damage to the endothelial glycocalyx seems to be comparable or even less
pronounced than in similar human settings. At the same time, the data from experiments
where current strategies, such as PCMV/PRV elimination, non-ischemic preservation and
growth inhibition, could not be applied indicate that damage to the endothelial glycocalyx
also plays an important role in cardiac xenotransplantation.
Author Contributions: Conceptualization, M.B., M.B.M. and M.L. (Matthias Längin); Data curation,
M.B., M.B.M. and M.L. (Matthias Längin); Formal analysis, M.B., M.B.M. and M.L. (
Matthias Längin
);
Funding acquisition, J.-M.A., B.R., E.W., P.B. and M.L. (Matthias Längin); Investigation, M.B. and
M.B.M.; Methodology, M.B., J.-M.A., M.B.M. and M.L. (Matthias Längin); Project administration,
J.-M.A.
, B.R., C.H., E.W., M.S., P.B. and M.L. (Matthias Längin); Resources, M.L. (Maria Leuschen),
F.W., J.R., E.N., M.M., I.B., S.M., R.E., K.G., S.S., A.P., J.D., A.W.G., R.R.T., C.H., D.A., E.W., M.S.
and P.B.; Software, M.B., M.B.M. and M.L. (Matthias Längin); Supervision, J.-M.A., B.R. and M.L.
(
Matthias Längin
); Validation, M.B., M.B.M. and M.L. (Matthias Längin); Visualization, M.B. and
M.B.M.; Writing—original draft, M.B.; Writing—review and editing, J.D., M.B.M. and M.L.
(Matthias Längin). All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the German Research Foundation (Deutsche Forschungsge-
meinschaft, DFG) TRR 127 and—in part—by the Swiss National Science Foundation (CR-SII5_198577),
the Bavarian Research Foundation (AZ-1543-22) and the Leducq Foundation (23CVD01).
Institutional Review Board Statement: The animal study protocol was approved by the Institutional
Review Board (or Ethics Committee) of the Government of Upper Bavaria (Regierung von Oberbayern,
protocol code ROB-55.2-2532.Vet_02-14-184, date of approval: 9 February 2015).
Informed Consent Statement: Not applicable.
Data Availability Statement: The raw data supporting the conclusions of this article will be made
available by the authors on request.
Acknowledgments: The authors thank the German Primate Center and the Walter Brendel Center of
Experimental Medicine for support and provision of facilities, especially D. Merkus, M. Shakarami,
and all animal caretakers. They acknowledge K. Reiman for providing the anti-CD40 monoclonal
antibody for the experiments.
Conflicts of Interest: Jan-Michael Abicht, Bruno Reichart, Eckhard Wolf, Paolo Brenner and Matthias
Längin are founders of XTransplant GmbH. David Ayares is chief executive officer and chief scientific
officer of Revivicor, Inc. The funders had no role in the design of the study; in the collection, analyses,
or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
The other authors declare no conflicts of interest.
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