Near Complete Repair After Myocardial Infarction in Adult Mice by Altering the Inflammatory Response With Intramyocardial Injection of α-Gal Nanoparticles

Abstract

Background: Neonatal mice, but not older mice, can regenerate their hearts after myocardial-infarction (MI), a process mediated by pro-reparative macrophages. α-Gal nanoparticles applied to skin wounds in adult-mice bind the anti-Gal antibody, activate the complement cascade and generate complement chemotactic peptides that recruit pro-reparative macrophages which are further activated by these nanoparticles. The recruited macrophages decrease wound healing time by ~50%, restore the normal skin structure and prevent fibrosis and scar formation in mice.
Objectives: The objective of this study is to determine if α-gal nanoparticles injected into the reperfused myocardium after MI in adult-mice can induce myocardial repair that restores normal structure, similar to that observed in skin injuries.
Methods and Results: MI was induced by occluding the mid-portion of the left anterior descending (LAD) coronary artery for 30 min. Immediately following reperfusion, each mouse received two 10 μl injections of 100 μg α-gal nanoparticles in saline into the LAD territory ( n = 20), or saline for controls ( n = 10). Myocardial infarct size was measured by planimetry following Trichrome staining and macrophage recruitment by hematoxylin-eosin staining. Left ventricular (LV) function was measured by echocardiography. Control mice displayed peak macrophage infiltration at 4-days, whereas treated mice had a delayed peak macrophage infiltration at 7-days. At 28-days, control mice demonstrated large transmural infarcts with extensive scar formation and poor contractile function. In contrast, mice treated with α-gal nanoparticles demonstrated after 28-days a marked reduction in infarct size (~10-fold smaller), restoration of normal myocardium structure and contractile function.
Conclusions: Intramyocardial injection of α-gal nanoparticles post-MI in anti-Gal producing adult-mice results in near complete repair of the infarcted territory, with restoration of normal LV structure and contractile function. The mechanism responsible for this benefit likely involves alteration of the usual inflammatory response post-MI, as previously observed with regeneration of injured hearts in adult zebrafish, salamanders and neonatal mice.

Full text

ORIGINAL RESEARCH
published: 25 August 2021
doi: 10.3389/fcvm.2021.719160
Frontiers in Cardiovascular Medicine | www.frontiersin.org 1August 2021 | Volume 8 | Article 719160
Edited by:
James J. H. Chong,
The University of Sydney, Australia
Reviewed by:
Zoe Clayton,
Westmead Institute for Medical
Research, Australia
Yuri D’Alessandra,
Monzino Cardiology Center
(IRCCS), Italy
*Correspondence:
Uri Galili
uri.galili@rcn.com
Specialty section:
This article was submitted to
Cardiovascular Biologics and
Regenerative Medicine,
a section of the journal
Frontiers in Cardiovascular Medicine
Received: 02 June 2021
Accepted: 30 July 2021
Published: 25 August 2021
Citation:
Galili U, Zhu Z, Chen J, Goldufsky JW
and Schaer GL (2021) Near Complete
Repair After Myocardial Infarction in
Adult Mice by Altering the
Inflammatory Response With
Intramyocardial Injection of α-Gal
Nanoparticles.
Front. Cardiovasc. Med. 8:719160.
doi: 10.3389/fcvm.2021.719160
Near Complete Repair After
Myocardial Infarction in Adult Mice
by Altering the Inflammatory
Response With Intramyocardial
Injection of α-Gal Nanoparticles
Uri Galili 1
*, Zhongkai Zhu 2, Jiwang Chen 2, Josef W. Goldufsky 1and Gar y L. Schaer 1
1Department of Medicine, Rush University Medical Center, Chicago, IL, United States, 2Department of Medicine, University
of Illinois at Chicago, Chicago, IL, United States
Background: Neonatal mice, but not older mice, can regenerate their hearts after
myocardial-infarction (MI), a process mediated by pro-reparative macrophages. α-Gal
nanoparticles applied to skin wounds in adult-mice bind the anti-Gal antibody, activate
the complement cascade and generate complement chemotactic peptides that recruit
pro-reparative macrophages which are further activated by these nanoparticles. The
recruited macrophages decrease wound healing time by ∼50%, restore the normal skin
structure and prevent fibrosis and scar formation in mice.
Objectives: The objective of this study is to determine if α-gal nanoparticles injected
into the reperfused myocardium after MI in adult-mice can induce myocardial repair that
restores normal structure, similar to that observed in skin injuries.
Methods and Results: MI was induced by occluding the mid-portion of the left
anterior descending (LAD) coronary artery for 30 min. Immediately following reperfusion,
each mouse received two 10 µl injections of 100 µgα-gal nanoparticles in saline
into the LAD territory (n=20), or saline for controls (n=10). Myocardial infarct
size was measured by planimetry following Trichrome staining and macrophage
recruitment by hematoxylin-eosin staining. Left ventricular (LV) function was measured
by echocardiography. Control mice displayed peak macrophage infiltration at 4-days,
whereas treated mice had a delayed peak macrophage infiltration at 7-days. At 28-days,
control mice demonstrated large transmural infarcts with extensive scar formation and
poor contractile function. In contrast, mice treated with α-gal nanoparticles demonstrated
after 28-days a marked reduction in infarct size (∼10-fold smaller), restoration of normal
myocardium structure and contractile function.
Conclusions: Intramyocardial injection of α-gal nanoparticles post-MI in anti-Gal
producing adult-mice results in near complete repair of the infarcted territory, with
restoration of normal LV structure and contractile function. The mechanism responsible
for this benefit likely involves alteration of the usual inflammatory response post-MI, as
previously observed with regeneration of injured hearts in adult zebrafish, salamanders
and neonatal mice.
Keywords: myocardial infarction, myocardial repair, mice, macrophages, anti-Gal antibody, α-gal epitope, α-gal
nanoparticles

Galili et al. α-Gal Nanoparticles Reduce Infarct Size
INTRODUCTION
Myocardial infarction (MI) is the cause of 25% of the deaths in
the USA, primarily because of the extremely limited regenerative
capacity of the myocardium (1). Myocardial damage post-MI
usually heals by the default repair mechanism of fibrosis and
scar formation which prevents subsequent rupture of the injured
ventricular wall. This repair mechanism often results in reduced
contractility which can lead to heart failure and premature death
(2). Several studies of repair after MI in mice have suggested that
this repair and healing mechanism is similar to that mediating
healing and scar formation in skin wounds (3–8). In both healing
events, “pro-inflammatory” polarized macrophages are the first
to reach the injury and debride it of dead cells. Subsequently,
“pro-reparative” polarized macrophages secrete cytokines which
orchestrate angiogenesis, fibrosis and scar formation. In contrast
to this repair mechanism, several vertebrates were found capable
of natural regeneration of the injured myocardium, thereby
restoring the original structure of the tissue without fibrosis.
These include adult zebrafish (9), adult amphibians, such as
salamander (10) and axolotl (11) and neonatal mice (12,13) and
pigs (14,15). If injury to the heart in these mammalian neonates
is caused during the first or second day after birth, the injured
myocardium regenerates into its original structure, whereas
injuries caused several days after birth result in fibrosis and scar
formation as in the adult animal. These myocardial regeneration
processes in fish, amphibians and mammalian neonates were
found to be associated with extensive infiltration of macrophages
into the injured tissue (16–20) and activation of the complement
system (20–23). We have aimed to determine whether it is
possible to induce by immunological means, extensive activation
of the complement system and recruitment of macrophages
into injured myocardium of post-MI adult mice, in order to
induce myocardial repair similar to that observed in adult fish,
amphibians and neonatal mice.
We have previously reported that extensive immune mediated
complement activation which results in macrophage recruitment,
is associated with accelerated regeneration and prevention
of fibrosis in skin injuries of adult mice treated with α-
gal nanoparticles (24–29). These nanoparticles present a
carbohydrate antigen, called the “α-gal epitope,” with the
structure Galα1-3Galβ1-4GlcNAc-R (25,27). α-Gal epitopes bind
the natural anti-Gal antibody which is abundant in all humans
and constitutes as much as ∼1% of immunoglobulins (30–33).
All mammals that are not monkeys or apes synthesize
the α-gal epitope. Among primates, lemurs (evolved in
Madagascar) and New World monkeys (monkeys of South
America) also synthesize the α-gal epitope (30,34,35). All
mammals synthesizing α-gal epitopes cannot produce the anti-
Gal antibody since the α-gal epitope is a self-antigen in
them. In contrast, humans, apes and Old-World monkeys
Abbreviations: α-gal, carbohydrate antigen with the structure Galα1-3Galα1-
4GlcNAc-R; BrdU, bromodeoxy uridine; ECM, extracellular matrix; FS, fractional
shortening; GT-KO, mouse or pig knockout for the α1,3galactosyltransferase
gene (GTTA1); LAD, left anterior descending; LV, left ventricle; MI, myocardial
infarction; PCNA, proliferating cell nuclear antigen; RBC, red blood cells.
(monkeys of Asia and Africa) lack α-gal epitopes and produce
the natural anti-Gal antibody without active immunization, in
response to constant antigenic stimulation by gastrointestinal
bacteria (30–36). The reason for these differences in α-gal
epitope synthesis in mammals is the differential activity of the
α1,3galactosyltransferase (α1,3GT) gene (also called GGTA1)
which codes the α1,3GT enzyme that synthesizes α-gal epitopes.
This gene is active in all mammals synthesizing α-gal epitopes
but has been evolutionary inactivated in ancestral apes and
Old-World monkeys, thus it is inactivated in humans, as
well (34,35,37).
Since anti-Gal is present in all humans and anti-Gal/α-gal
immune complexes effectively activate the complement system,
we hypothesized that formation of such immune complexes
in the form of anti-Gal interaction with α-gal nanoparticles
may be considered as a platform for future induction of a
variety of regenerative therapies (30). The previous studies
on skin injury repair and regeneration (24–29) indicated that
anti-Gal/α-gal nanoparticles interaction at the administration
site of these nanoparticles, activates the complement system to
generate large amounts of C5a and C3a complement cleavage
peptides that induce recruitment of multiple macrophages into
the treated injuries. The recruited macrophages bind via their Fc
receptors the Fc “tail” of anti-Gal immunocomplexed with the
multiple α-gal epitopes on the nanoparticles and are activated to
polarize into pro-reparative macrophages that secrete a variety of
cytokines which decrease the healing time by ∼50% and prevent
fibrosis and scar formation.
In the present study we hypothesized that α-gal nanoparticles
may contribute to repair of post-MI mouse heart and prevent
scar formation, as illustrated in Figure 1. The various stages of
the repair process, as hypothesized in adult mouse heart, are
as follows: Stage 1. Post-MI injection of α-gal nanoparticles
into the injured myocardium of mice producing anti-Gal will
result in anti-Gal/α-gal nanoparticles interaction which activates
the complement system to generate the complement cleavage
chemotactic peptides C5a and C3a that recruit macrophages.
Stage 2. Recruited macrophages bind via their Fc-receptors the Fc
“tail” of anti-Gal coating the α-gal nanoparticles and are induced
to polarize into macrophages secreting pro-reparative cytokines.
Stage 3. The pro-reparative cytokines induce restoration of
structure and function of the injured myocardium.
The study of this hypothesis required a unique strain
of knockout mice (called GT-KO mice) in which the
α1,3galactosyltransferase gene (α1,3GT gene also called
GGTA1) is disrupted (38). GT-KO mice do not produce anti-Gal
unless they are immunized with immunogenic glycoproteins
(e.g., xenoglycoproteins) presenting multiple α-gal epitopes. The
immunogenic glycoproteins activate helper T cells which cannot
be activated by α-gal epitopes (39). Pig kidney membrane (PKM)
homogenate of wild-type pigs were used as an immunogen for
this purpose since glycoproteins in these cell membranes present
large amounts of α-gal epitopes (39–41). The mice require such
immunization for producing anti-Gal because they live in a
sterile environment that does not enable the development of a
gastrointestinal flora, which in humans induces production of
the natural anti-Gal antibody (25,30,31,39). MI was performed
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Galili et al. α-Gal Nanoparticles Reduce Infarct Size
FIGURE 1 | Hypothesis on post-MI myocardial repair by intramyocardial
injection of α-gal nanoparticles: Stage 1. Anti-Gal binding to injected α-gal
nanoparticles activates the complement system to generate chemotactic
peptides that recruit macrophages. Stage 2. Recruited macrophages bind via
Fc-receptors the Fc “tail” of anti-Gal coating the α-gal nanoparticles and are
induced to polarize into macrophages secreting pro-reparative cytokines.
Stage 3. The pro-reparative cytokines induce structure and function
restoration of the injured myocardium.
in the mice by an occlusion/reperfusion procedure. Here we
demonstrated near complete repair of the ischemia injured
myocardium following intramyocardial injection of α-gal
nanoparticles in post-MI adult mouse hearts.
METHODS
Preparation of α-Gal Nanoparticles
α-Gal nanoparticles were prepared as previously described
(24,25,27) from rabbit red blood cell (RBC) membranes
because these RBC present the highest concentration of α-
gal epitopes in comparison to other mammalian RBC and
a large proportion of these α-gal epitopes is presented on
glycolipids (34). Rabbit RBC (1 L) are lysed in water and
washed for hemoglobin removal. Washed RBC membranes
are mixed with 800 ml chloroform and 800 ml methanol for
2 h, then 800 ml methanol are added and stirred overnight,
resulting in the extraction of the phospholipids, cholesterol and
glycolipids into the chloroform:methanol solution (24,25,40).
Residual RBC membranes and proteins are precipitated by the
chloroform:methanol solution. The precipitate is removed by
filtration through Whatman paper. The extract is dried in a rotary
evaporator, weighed, and sonicated in saline, in a sonication bath
to generate a suspension of 100 mg/ml liposomes comprised
of phospholipids, cholesterol and glycolipids. Residual debris is
pelleted at low speed centrifugation (800 rpm) and removed.
Liposomes are further sonicated on ice with a sonication
probe to break the liposomes into submicroscopic liposomes
(50–300 nm), referred to as “α-gal nanoparticles,” which are
sterilized by filtration through a 0.45 µm filter (Millipore).
These nanoparticles present ∼1015 α-gal epitopes per mg
nanoparticles (25). Nanoparticles lacking α-gal epitopes were
produced by the same method from RBC of knockout pigs
for the α1,3galactosyltransferas gene (GT-KO pigs) that cannot
synthesize α-gal epitopes (42,43).
Mouse Experimental Model
The experimental protocol was approved by the Institutional
Animal Care and Use Committee at Rush University Medical
Center (Chicago, IL) and performed in accordance with
AAALAC guidelines. In order to simulate a human-like immune
environment, a previously established, α1,3galactosyltransferase
(α1,3GT) knockout mouse (GT-KO) was used (38). Like humans,
these knockout mice do not synthesize the α-gal epitope and
therefore can produce the anti-Gal antibody with post-natal
exposure to this epitope, such as immunization with PKM
homogenate (24,25,39,40). PKM were empirically found to
be more effective than rabbit RBC in immunization of GT-KO
mice for the production of the anti-Gal antibody (unpublished
observations). GT-KO mice used (males and females, age 12–
16 weeks) were of C57BL/6 ×BALB/c genetic background (38).
Mice were induced to produce the anti-Gal antibody at titers
comparable to those in humans by 5 weekly immunizations with
50 mg PKM homogenate (200 mg/ml). These homogenates were
used for immunization that elicits anti-Gal production because
pig kidney membranes present a large number of α-gal epitopes
(41). In the absence of such immunization, GT-KO mice do not
produce anti-Gal because of lack of immunizing gastrointestinal
bacteria (39).
Induction of MI and Injection of α-Gal
Nanoparticles
MI induction with subsequent coronary reperfusion was
performed in mice based on modification of previously published
protocols (44,45). Mice were anesthetized with ketamine (100
mg/kg)/xylazine (5 mg/kg) injected intraperitoneal, intubated
and anesthesia maintained by inhaled 1.0% isoflurane. A left
thoracotomy was performed via the fourth intercostal space
and lungs were retracted to expose the heart. The mid-LAD
coronary artery was ligated with a 7-0 silk suture. Ligation was
confirmed as 100% occlusive by the appearance of pallor of the
anterior wall of the left ventricle (LV). After 30min occlusion,
the ligature was removed, permitting reperfusion of the LAD
territory. This was confirmed by noting a change in the color of
the anterior wall of the LV from pallor to deep red, as observed
prior to coronary occlusion. One minute after reperfusion, a
32-gauge needle was used to inject 10 µl of a 10 mg/ml α-
gal nanoparticles suspension in saline [dose optimized in refs.
(24,25)] into the LV anterior wall, ∼1 mm distal to the LAD
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Galili et al. α-Gal Nanoparticles Reduce Infarct Size
ligation site. Treated mice received two injections, 1–2 mm apart
(100 µg nanoparticles per site). Control mice underwent the
same procedure but received two injections of 10 µl normal
saline. Subsequently, the chest wall, subcutaneous tissue, and
skin were sutured. Among treated animals, 20 underwent post-
mortem studies on day 28 post-MI, and 2 animals per day at 4, 7,
and 14 days post-MI. Among controls, 10 mice underwent post-
mortem studies on day 28 post-MI, and 2 animals per day, 4, 7,
and 14 days post-MI. Mice were euthanized by CO2inhalation
followed by cervical translocation.
Histologic Specimen Preparation
Formalin fixed hearts were cut from mid-level (at the level
of the LAD occlusion) into 5 equal double sections (5 µm-
thick) 300 µm apart. Sections at each level were stained
with either hematoxylin-eosin (H&E) (to assess macrophage
infiltration), or Trichrome (collagen/fibrosis stain blue; viable
cardiomyocytes stain red; debrided cardiomyocyte areas stain
gray, RBCs stain brown). The LV section demonstrating the
greatest extent of macrophage infiltration or the greatest infarct
size (out of the sections prepared at 5 levels), was scanned
for planimetry measurement (46) using AperioImageScope
planimetry program. The scanned histology slides were measured
for infarct size in a blinded manner according to a code number
which subsequently was compared with a key list which indicated
the treatment of each heart. The infarct size for each scanned
section was expressed as percentage, relative to the total area of
uninjured LV (46). Sections were also stained with antibody to
F4/80 antigen for identification of macrophages (25) and with
antibodies to proliferating cell nuclear antigen (PCNA) (Abcam,
Cambridge, MA) for detection of proliferating cardiomyocytes.
The staining was performed following antigen retrieval with Tris-
EDTA buffer (10 mM Tris Base, 1 mM EDTA solution, 0.05%
Tween 20, pH 9.0, kept for 10 min in boiling solution) and
completed with a fluorescein coupled secondary antibody. After
washes the slides were counterstained with DAPI.
Assessment of LV Function With
Transthoracic Echocardiography
High resolution transthoracic echocardiography (Vevo 770,
VisualSonics) was performed in mice (n=4 treated, n=4
controls) that were sedated with 1% isoflurane, before LAD
ligation, and post-MI (at 7 and 28 days). Parasternal short-axis
views at the mid-ventricular level were obtained for M-mode
analysis of the LV internal diameter at end diastole (LVDD) and
end systole (LVDS). Fractional shortening (FS) was calculated as:
%FS =[(LVDD – LVDS)/LVDD] ×100.
Staining for Proliferating Cells by Nuclear
BrdU Uptake
Mice were injected intraperitoneally with 1.0 ml of a solution
containing 1 mg BrdU (Sigma-Aldrich, St. Louis, MO). The
injection was performed on Day 9 post-MI or on Day 11 post-MI.
The mice were euthanized after 24 and 48 h, heart and intestine
were harvested fixed in formalin and embedded in paraffin. The
intestine was used as positive control for each of the hearts
studied. Paraffin sections were deparaffinized and subjected to
BrdU nuclear staining by the use of “BrdU in situ Detection Kit”
(BD Pharmingen cat. No. 550803), according to the manufacturer
instruction. The kit includes biotinylated anti-BrdU antibody
and avidin coupled peroxidase. The color reaction is achieved
by diaminobenzidine (DAB) precipitation. Counter staining was
performed with hematoxylin.
Statistics
The data were presented as mean ±SE. Comparisons between
2 groups were made using Student t-test. Multiple group
comparisons were made using One-way ANOVA, followed by
Tukey’s multiple comparison test.
RESULTS
Effects of Anti-Gal Interaction With α-Gal
Nanoparticles
Some of the outcomes of anti-Gal interaction with α-gal
nanoparticles, hypothesized in Stages 1 and 2 in Figure 1 are
illustrated in Figure 2. Anti-Gal within the serum of GT-KO
mice producing this antibody readily binds to α-gal epitopes on
α-gal nanoparticles, as shown by flow cytometry in Figure 2A.
Nanoparticles lacking α-gal epitopes (produced from RBC of GT-
KO pigs that lack these epitopes) do not bind anti-Gal produced
by GT-KO mice. The binding of anti-Gal to α-gal nanoparticles
was previously shown to result in activation of the complement
system and production of complement cleavage chemotactic
peptides that effectively induce macrophage recruitment to
the nanoparticles injection sites in the uninjured skin of GT-
KO mice (25). A similar recruitment of macrophages could
be demonstrated by two injections (each of 10 µl of the α-
gal nanoparticles) into uninjured LV myocardium of healthy
hearts, not subjected to LAD occlusion. This is shown in a
representative heart evaluated 4 days post-injection (Figure 2B).
High power magnification inspection of the section in Figure 2B
and of the other 3 uninjured normal hearts that received
the same injection technique revealed macrophages and no
polymorphonuclear cells or fibroblasts among the infiltrating
cells. Similar infiltration of only macrophages (indicated as
F4/80 stained cells) was previously observed in mouse dermis
injected with α-gal nanoparticles (25,27). Injection of saline
into uninjured hearts resulted in no infiltration of macrophages
or other cells at any time point (not shown). The similarity in
recruitment of macrophages in uninjured skins (25) and hearts
injected with α-gal nanoparticles (Figure 2B), strongly suggests
that complement cleavage chemotactic peptides produced as
a result of anti-Gal/α-gal nanoparticles interaction, shown to
mediate recruitment of macrophages in the skin (25) have a
similar chemotactic effect in post-MI reperfused hearts injected
with these nanoparticles, as illustrated in Stage 1 of Figure 1.
The predicted Fc/Fc receptor interaction between anti-
Gal coated α-gal nanoparticles and recruited macrophages
(Stage 2 in Figure 1) is shown in Figure 2C with macrophages
incubated for 2 h at 24◦C with 10 mg/ml of such nanoparticles.
The representative macrophage is covered with the bound
α-gal nanoparticles that are coated with anti-Gal. The
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Galili et al. α-Gal Nanoparticles Reduce Infarct Size
FIGURE 2 | Characteristics of anti-Gal interaction with α-gal nanoparticles: (A) Flow cytometry evaluation of anti-Gal IgG binding to α-gal nanoparticles (blue)
(representing Stage 1 of Figure 1); nanoparticles lacking α-gal epitopes (red), isotype-control (gray), auto-fluorescence without antibodies (black). (B) In vivo
recruitment of macrophages by α-gal nanoparticles, 4 days post-injection of the nanoparticles into normal uninjured mouse heart (also representing Stage 1 of
Figure 1). The section is in a representative heart of 4 hearts with similar results. Arrows – macrophages infiltrating two injection sites. Staining by hematoxylin-eosin
(H&E). (C) Scanning electron microscopy of anti-Gal-coated α-gal nanoparticles binding to a macrophage (representing Stage 2 in Figure 1). (D) As (C) however the
initial concentration of the applied α-gal nanoparticles is 100-fold lower, thus the cell membrane of the macrophage is visible.
binding of anti-Gal coated α-gal nanoparticles also causes
the contraction of the macrophage into a more spherical
shape because of the multiple Fc/Fc receptor interactions.
When the nanoparticles concentration is 100-fold less (i.e.,
0.1 mg/ml) fewer nanoparticles bind to the macrophage, thus
the macrophage maintains a normal flattened morphology
(Figure 2D). In the absence of anti-Gal, no nanoparticles were
bound to the macrophages (not shown).
Histological Evidence of Myocardial Repair
With α-Gal Nanoparticles
The hypothesized Stage 3 in Figure 1 suggests that following the
Fc/Fc receptor interactions between the recruited macrophages
and anti-Gal coated α-gal nanoparticles in injured myocardium,
the macrophages polarize into pro-reparative macrophages that
secrete cytokines which, similar to their effects in wound
healing, enhance repair and restore the normal structure and
function of the post-MI myocardium. These hypothesized effects
were studied in GT-KO mouse hearts assessed 28 days after
the 30 min of mid-LAD occlusion and reperfusion in mice
treated with α-gal nanoparticles, and saline-treated controls.
The normal mouse heart (not subjected to LAD occlusion)
(Figure 3A) demonstrates the expected uniform Trichrome red
staining of uninjured cardiomyocytes and no fibrosis. Dashed
circles mark epicardial and endocardial surfaces of the LV.
The area between the circles represents 100% uninjured LV.
The right ventricle (when visible) is on the left, and the LV
on the right of each figure. The size of myocardial infarct
(Trichrome staining collagen/fibrosis blue) was calculated as
percentage, relative to the area of uninjured LV, based on
planimetry measurements (46) of the injured and non-injured
LV (Figure 3B). The histological sections of hearts providing
the data for Figure 3B are presented in Figures 3C,D and in
Supplementary Figure 1.
In saline injected controls (Figure 3C;
Supplementary Figure 1A), mid-LAD occlusion for 30 min
followed by reperfusion for 28 days resulted in large transmural
infarcts with extensive fibrosis, scar formation and wall thinning.
The infarct size, extent of fibrosis and patterns of scar formation
are highly variable among the 10 control animals, in which
the % mean ±SE infarct size of the LV was 19 ±2.3%, with a
range of 5–35% - (Figure 3B). This variability may result from
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Galili et al. α-Gal Nanoparticles Reduce Infarct Size
FIGURE 3 | Myocardial repair 28 days post-MI in adult mice treated with α-gal nanoparticles. (A) Normal heart- dashed circles demarcate LV (Trichrome, uninjured
cardiomyocytes-red, RBCs-brown). (B) Infarcted size determined by fibrosis due to myocardial infarction, measured by planimetry and calculated as percentage,
relative to the area of uninjured LV in 10 saline controls, 20 α-gal nanoparticles treated hearts, 3 hearts injected with nanoparticles lacking α-gal epitopes, and 3 hearts
from mice lacking anti-Gal and treated with α-gal nanoparticles. Results are also presented as mean (dashed lines) ±SE. Horizontal solid lines indicate statistical
comparisons between groups. Statistical analysis was performed by One-way ANOVA, followed by Tukey’s multiple comparison test, ****p<0.0001;
***p<0.001. (C–E) Trichrome staining (collagen/fibrosis stains-blue, uninjured cardiomyocytes-red, RBCs-brown). (C) Saline treated hearts (additional 5 in
Supplementary Figure 1A). Fibrosis areas magnified in lower figure of each pair. (D) α-Gal nanoparticles treated hearts (additional 15 in Supplementary Figure 1B).
Fibrosis areas magnified in lower figure of each pair. (E) Border between fibrotic tissue and healthy appearing cardiomyocytes in α-gal nanoparticles treated hearts. (F)
H&E staining of E, demonstrating sarcomeric striation within cardiomyocytes of normal appearance [in (E,F), one representative of 10 hearts with similar results].
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Galili et al. α-Gal Nanoparticles Reduce Infarct Size
small differences in the site of LAD occlusion and amount of
myocardium supplied distal to the occlusion. Similar distribution
of infarct size following 30 min occlusion/reperfusion in mice
also was observed by other investigators (47–49). Twenty mice
treated with α-gal nanoparticles demonstrated dramatically
smaller infarcts, with greatly less fibrosis and thinning of the
LV wall (Figure 3D;Supplementary Figure 1B). The mean
myocardial infarct size in treated animals was 2.2 ±1.2%,
with a range of 0.1–5.0% (Figure 3B). The tissue stained
red surrounding the residual blue fibrotic tissue in α-gal
nanoparticles treated hearts (Figure 3E) is comprised of
normal appearing cardiomyocytes that display (under high
magnification of H&E stain) characteristic sarcomeric striation
(Figure 3F), with minimal fibrosis.
Two additional types of controls demonstrate that the
reparative effects of α-gal nanoparticles are dependent on the
specific interaction between the anti-Gal antibody and α-gal
epitopes on the nanoparticles, as hypothesized in Figure 1.
Injection of GT-KO pig nanoparticles (i.e., nanoparticles lacking
α-gal epitopes) into the reperfused post-MI myocardium resulted
in fibrosis and scar formation that is similar to mice treated
with saline (Figure 3B). The individual hearts receiving this
treatment are shown in Supplementary Figure 2A. Furthermore,
injection of α-gal nanoparticles into the post-MI heart of GT-
KO mice lacking anti-Gal (i.e., not immunized with pig kidney
membranes homogenate) resulted in fibrosis and scar formation,
as well (Figure 3B;Supplementary Figure 2B), rather than
in extensive myocardial restoration of structure, observed
in anti-Gal producing mice. Thus, both anti-Gal production
and α-gal epitopes on the nanoparticles are required for the
induction of the reparative response in injured myocardium of
adult mice.
Evidence of Restoration of LV Function
With α-Gal Nanoparticles
Echocardiography was used to assess LV function in four saline
treated controls (Figure 4A) and four α-gal nanoparticles treated
mice (Figure 4B). Mice in both groups had normal fractional
shortening (FS) before LAD occlusion (Figures 4C,D). By Day
7 post-MI, both groups demonstrated a marked reduction
in FS, associated with LV chamber dilation, emphasizing the
severity of the ischemic injury (Figures 4C–E). These Day 7
echocardiography results indicate that the post-ischemia injuries
in both saline and α-gal nanoparticles treated hearts were severe
and correlate with the histopathology results demonstrated below
in Figure 5.
However, by Day 28, α-gal nanoparticles treated mice
demonstrated a dramatic recovery of FS and return of LV
chamber dimension to normal size, whereas control mice
continued to display impaired FS and chamber dilation
(Figures 4C–E). It is of note that the mice included in Figure 4
also provided histopathology results at Day 28. These results are
included among the 10 saline treated mice which demonstrated
fibrosis and scar formation and among 20 α-gal nanoparticles
treated mice demonstrating repair and restoration of structure
(Figure 3;Supplementary Figure 1) and are consistent with the
functional results in Figure 4.
α-Gal Nanoparticles Alter the Inflammatory
Response Post-MI
The near-complete restoration of structure and function in
the post-MI myocardium treated with α-gal nanoparticles,
compared to the extensive fibrosis and scar formation in
saline injected control hearts, raised the question whether
there are observable differences in the time-course of the
inflammatory response in the two groups. This was assessed
by histopathology at 4, 7, and 14 days post-MI (Figure 5).
Myocardial histopathology in one of two mice studied at
each time point is shown in pairs of which the upper is
stained with H&E and lower with Trichrome. In these sections,
H&E staining identifies the infiltrating macrophages which are
much smaller than the cardiomyocytes and lack the elongated
shape and sarcomere striation, whereas Trichrome staining
identifies areas debrided of injured cardiomyocytes (stained
gray) and areas containing healthy cardiomyocytes (stained
red). Planimetry measurements of the debrided areas in two
mice at each time point are presented in Figure 5C and the
number of observed macrophages in Figure 5D. The identity
of the infiltrating cells as macrophages is demonstrated in
Figures 5E,F by the staining with the macrophage specific
F4/80 antibody.
At Day 4, the myocardium of control animals displayed
extensive cardiomyocytes elimination (gray staining with
Trichrome), coinciding with extensive infiltration of
macrophages, similar to previous reports (4–8,50–52).
Hearts treated with α-gal nanoparticles displayed distinctly
less macrophage infiltration at Day 4 (Figures 5B–D), suggesting
an attenuated early inflammatory response. In accord with
previous observations (52), the number of macrophages in
control hearts greatly decreased within the injured myocardium
at Day 7, whereas at Day 14, there was thinning and fibrosis of
the LV wall and very low macrophage content (Figures 5A,C,D).
In contrast to control mice, α-gal nanoparticles treated hearts
demonstrated at Day 7 a marked increase in macrophage
infiltration in the area debrided of cardiomyocytes. The areas
in which cardiomyocytes died and were debrided in these mice
are stained gray in Trichrome staining and comprise 19–24%
of the LV myocardium (Figures 5B,C). The infiltrating cells on
Day 7 were confirmed to be macrophages by positive in situ
staining of the infiltrating mononuclear cells (Figure 5E, H&E)
with the macrophage specific peroxidase-linked F4/80 antibody
(Figure 5F). The areas in which macrophages infiltrated at Day
7 in the α-gal nanoparticles treated hearts, fully overlapped the
areas devoid of cardiomyocytes (Figure 5B). This is further
demonstrated in additional sequential sections at different planes
of Day 7 α-gal nanoparticles treated heart (Figure 5G, middle
section also shown in Figure 5B). Histological sections above
and below the middle section (left and right figures, respectively)
display a different distribution of infiltrating macrophages,
suggesting that the injured area is of a substantial size with
three-dimensional irregular shape. The overlap in the shape
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Galili et al. α-Gal Nanoparticles Reduce Infarct Size
FIGURE 4 | Echocardiography of (A) 4 saline controls (a1–4), and (B) 4α-gal nanoparticles treated mice (b1–4), pre-LAD ligation, 7- and 28-days post-ligation. (C)
Fractional-shortening (FS) at each 3 time points. Dashed-lines and open-circles represent control mice (a1–4), solid-lines and closed-circles represent treated mice
(b1–4). (D) Mean ±SE at each time points. FS data for the two groups are significantly different only on Day 28. Statistical analysis was performed by Student t-test, p
<0.001. (E) Data presented as percentage changes from baseline in Fractional Shortening at 7- and 28-days post-ligation. Symbols and lines are as in (C).
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Galili et al. α-Gal Nanoparticles Reduce Infarct Size
of the macrophage infiltrate (H&E) and the area devoid of
cardiomyocytes (gray color in Trichrome staining) in Figure 5G,
further suggests that a high proportion of the cardiomyocytes
died following the ischemia and that one of the activities of
macrophages recruited by α-gal nanoparticles is debriding areas
of necrotic cardiomyocytes that were injured by the 30 min of
LAD occlusion. Similar to control mice, very few macrophages
were detected at Day 14 in α-gal nanoparticles treated hearts
(Figures 5B,D). However, in clear contrast to control hearts
which displayed at Day 14 thinning of the LV wall and fibrosis,
α-gal nanoparticles treated hearts at Day 14 displayed near-
complete restoration of myocardium structure and only minor
residual fibrosis (Figures 5B,C). These observations strongly
suggest that the areas debrided of cardiomyocytes at Day 7
in α-gal nanoparticles treated hearts were repopulated with
healthy cardiomyocytes, as shown in hearts studied both at
Days 14 and 28. It is of note that although the kinetics of
macrophage infiltration differs between saline treated hearts
and α-gal nanoparticles treated hearts, the damage caused
to the myocardium (i.e., % infarct size) is not significantly
different in both groups on Day 7. This observation is supported
by the echocardiography studies displaying similar impaired
contractility in the two groups on Day 7 (Figure 4). However,
by Day 28 the contractility is restored to normal in the α-
gal nanoparticles treated mice vs. continuation in impaired
contractility in saline treated mice.
Evaluating Cell Proliferation in α-Gal
Nanoparticles Treated Hearts
The histological studies on post-MI myocardial repair presented
in Figure 5 imply that the repopulation by cardiomyocytes
following injection of α-gal nanoparticles occurs in the
second week post-treatment. This raised the question whether
the repopulation of the injured myocardium with healthy
cardiomyocytes is the result of proliferation of pre-existing
cardiomyocytes, or of resident stem/progenitor cells. This was
studied on Days 9 and 11 post-MI in mice treated with α-gal
nanoparticles by evaluating BrdU (thymidine analog) uptake
into nuclei of proliferating cells in the heart 24 and 48 h after
intraperitoneal injection of 1 mg BrdU. The sections were stained
overnight with the biotinylated anti-BrdU antibody followed by
avidin-peroxidase and DAB precipitation. Figure 6 shows the
results in a representative mouse studied 24 h post-injection
of BrdU on Day 9 (out of 5 mice with similar results). BrdU
was effectively taken up by nuclei of proliferating cells at
the base (crypt) of intestinal villi of the mice, which served
as positive controls of proliferating epithelium (Figure 6A).
However, in the heart, the staining of nuclei was sparse and
mostly of the stained nuclei that did not correspond to distinct
cardiomyocytes (Figures 6B,C). In view of distinct areas of
injured cardiomyocytes debrided by macrophages (Figure 5), it
would be expected that repopulation of these debrided areas by
cells proliferating within the heart will result in appearance of
groups of cardiomyocytes displaying uptake of BrdU. Similarly,
no such BrdU staining pattern has been observed in any of
the hearts in mice injected with BrdU on Day 9 and evaluated
48 h later, or in mice injected on Day 11 and evaluated after
24 or 48 h (not shown). In addition, no distinct nuclear BrdU
staining was detected in cardiomyocytes in regions adjacent to
areas containing infiltrating macrophages (Figure 6D).
A second method for detecting proliferating cardiomyocytes
was performed by anti-PCNA antibody staining of sections of
treated hearts harvested on Days 9 and 11. This antibody binds
to the proliferating cell nuclear antigen (PCNA) which is a DNA
clamp found in nuclei at the stage of DNA synthesis. As shown
in Figure 7A, nuclei of proliferating cells are readily stained by
anti-PCNA at the base (crypts) of intestinal villi. In contrast, no
distinct staining of cardiomyocyte nuclei was observed in the
hearts treated with α-gal nanoparticles and harvested 9 or 11 days
post-MI (Figures 7B,C). However, in view of the staining of a
small number of nuclei observed in treated hearts both by BrdU
uptake (Figures 6B,C) and by staining for PCNA (Figures 7B,C),
we cannot regard these observations as conclusive evidence that
there is a complete absence of proliferating cells in post-MI hearts
treated with α-gal nanoparticles.
DISCUSSION
The present study demonstrates near-complete myocardial repair
of the infarcted territory in anti-Gal producing adult GT-
KO mice subjected to 30 min of LAD occlusion, followed
by intramyocardial injection of α-gal nanoparticles shortly
after reperfusion. In marked contrast, saline-injected controls
demonstrate much larger infarcts, extensive myocardial thinning,
fibrosis and scar formation in the infarct territory with
corresponding impairment of LV function and persistent LV
dilation. Natural regeneration of injured myocardium is observed
in adult zebrafish (9) and amphibians (10,11), whereas in mice
and pigs it is observed in neonates only for a day or two after
birth (12–15). The temporary post-natal natural regeneration in
these mammals has led to the assumption that the mammalian
heart retains a regenerative capacity, but the molecular switches
are shut off in neonates shortly after birth (53,54). Thus, it
has been suggested that restoring the myocardial regenerative
response in post-MI adult mammals would require “turning back
the cardiac regenerative clock” (54). Presently, the mechanism
of myocardium repair in post-MI adult mice treated with α-
gal nanoparticles, is far from being understood. However, the
similarities between natural regeneration in fish, amphibians and
neonatal mice and the repair following the α-gal nanoparticles
treatment in adult mice may provide some suggestions regarding
this mechanism.
Activation of the complement system is common to the
natural regeneration and repair induced by α-gal nanoparticles.
Natural regeneration studies reported that activation of the
complement cascade and upregulation of complement receptors
in cells at the injury site are phenomena observed in
adult zebrafish, amphibians and neonatal mice with various
injuries, including heart injuries (22). Accordingly, activation of
complement C5a receptor1 (C5aR1) and of C3aR1 mediates an
evolutionarily conserved response that promotes cardiomyocyte
proliferation after cardiac injury in all these vertebrates
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Galili et al. α-Gal Nanoparticles Reduce Infarct Size
displaying natural regeneration (23). Moreover, regeneration of
injured hearts was reduced in neonatal mice with blocking of C5a
binding to its receptor C5aR1 or following genetic deletion of
C5aR1 (23). It is not clear as yet what is the signal that activates
the complement cascade in the course of natural regeneration,
and which is absent in adult mice. It is probable, however, that
activation of the complement cascade in neonates is not the
result of any antigen/antibody interaction because the injury
(e.g., clipping the apex of the heart) does not introduce new
foreign antigens. Nevertheless, it is well-established that in anti-
Gal producing adult mice treated with α-gal nanoparticles, there
is an extensive activation of the complement cascade following
anti-Gal binding to α-gal nanoparticles (25). The complement
cleavage peptides produced by complement activation due to
this antigen/antibody interaction, may trigger mechanisms for
myocardium regeneration, similar to those mediating natural
regeneration in neonatal mice.
A second characteristic common to natural injured heart
regeneration and to post-MI myocardial repair following
α-gal nanoparticles treatment is the extensive recruitment
of macrophages into the injured myocardium (16–20).
Nevertheless, extensive macrophages infiltration is also
observed in untreated (control) post-MI hearts in adult
mice (4–6,51,55,56). Both mouse macrophages infiltrating into
untreated post-MI hearts (51,55) and macrophages recruited
by α-gal nanoparticles in sponge disc implants (29) were found
to display M2 polarization. In view of these observations, the
polarization state of the infiltrating macrophages in post-MI
hearts treated with by α-gal nanoparticles was not determined.
Finding of M2 polarization in the latter hearts will not help
in differentiating between macrophages mediating repair in
the present study and those which mediated fibrosis and scar
formation in untreated or saline treated hearts. In addition,
there are increasing concerns that M1/M2 staining in post-MI
heart histopathology may not be useful in characterization of
macrophages in post-MI heart in mice (57).
Our studies have indicated however, that there are two
distinct differences between these two macrophage populations:
1. Macrophages infiltrating the treated myocardium are recruited
by chemotactic complement cleavage peptides such as C5a
and C3a generated by anti-Gal interaction with the α-gal
nanoparticles (Stage 1 in Figure 1). If the complement system is
inactivated, no such recruitment is observed in skin injected with
α-gal nanoparticles (25). Accordingly, macrophage recruitment
is also observed in uninjured myocardium injected with α-gal
nanoparticles (Figure 2B). Peak macrophage infiltration in
injured hearts injected with α-gal nanoparticles is observed at
Day 7 (Figure 5B). In contrast, in control saline injected post-MI
myocardium, peak infiltration of macrophages is at Day 4 and
the recruitment of these macrophages is mediated by substances
released from ischemic myocardium such as Monocyte
Chemoattractant Protein 1 (MCP-1) (58,59) (Figure 5A). 2.
Macrophages infiltrating the treated myocardium bind anti-
Gal coated α-gal nanoparticles via Fc/Fc receptor interaction
(Stage 2 in Figure 1), as shown in Figures 2C,D, whereas these
nanoparticles are absent in the control post-MI myocardium.
It is well-established that this Fc/Fc receptor interaction results
FIGURE 5 | Post-MI injury and macrophage infiltration at 4, 7, 14 days
post-MI in controls and treated mice (n=2 at each time point). In (A,B), the
(Continued)
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Galili et al. α-Gal Nanoparticles Reduce Infarct Size
FIGURE 5 | upper image of each pair is stained with H&E (arrows indicate areas of macrophage infiltration); lower image is stained with Trichrome (areas debrided of
cardiomyocytes are stained gray). (A) One representative saline control at each of 3 time points post-MI. (B) One representative α-gal nanoparticles treated animal at
each time point. The arrows mark macrophages infiltrating areas. Note on Day 4, arrows mark the macrophages at the two nanoparticles injection sites. (C)
Planimetry of injured myocardium as percent of LV relative to the area of uninjured LV in two hearts per time-point. Open-columns: saline control: closed-columns:
α-gal nanoparticles treatment. (D) Quantification of infiltrating macrophages within injured myocardium (H&E), columns as in (C).(E) Staining of the infiltrating cells into
heart injected with α-gal nanoparticles and viewed on Day 7 post-MI (H&E). (F) Cells in (E) are macrophages as they stain with peroxidase coupled anti-F4/80
antibody, an antibody that binds specifically to macrophages (Representative of three hearts with similar results). (G) Demonstration of the full overlap between the
area with infiltrating macrophages (H&E staining) and the corresponding area debrided of injured cardiomyocytes (Trichrome staining). The sections to the left and right
of the middle section (also shown at Day 7, B) are 300 µm above and below the middle section, respectively. The full overlap between areas of macrophages and
debrided areas strongly suggests that the macrophages debride the damaged cardiomyocytes in the areas they reside.
FIGURE 6 | Staining for BrdU uptake by proliferating cells, 24 h following intraperitoneal injection of 1 mg BrdU. Injection was performed on Day 9 post-MI and
intramyocardial α-gal nanoparticles injection. (A) Intestinal villi demonstrating effective uptake of BrdU in proliferating cells at the base of the villi (serving as a positive
control). (B–D) Staining of the LV for BrdU uptake in an α-gal nanoparticles treated mouse. Only sparse uptake is observed. In (D) the section displays an area of
macrophage infiltration near the cardiomyocytes. Data from one mouse of 5 with similar results.
in activation of multiple genes in macrophages, including a
number of genes encoding a wide variety of cytokines (60,61).
Accordingly, interaction of recruited macrophages with anti-Gal
coated α-gal nanoparticles was found to produce cytokines such
as VEGF, IL1α, FGF, PDGF and CSF (25,27). Thus, it is probable
that macrophages interacting with α-gal nanoparticles within
the post-MI injured myocardium also produce cytokines that
mediate repair of the treated myocardium. Such cytokines may
be absent in the control post-MI injured myocardium, ultimately
resulting in the default fibrosis rather than restoration of the
normal structure of the myocardium.
One of the unsolved questions emerging from the present
study is the nature of the extensive cellular repair with
cardiomyocytes in the debrided regions, occurring between Days
7 and 14 in treated post-MI hearts (Figures 5B,C). Histological
examination suggests an intensive repopulation of the necrotic
myocardium with healthy cardiomyocytes without evidence
of cardiomyocyte hypertrophy (Figure 3F). Cardiomyocyte
proliferation under the effect of cytokines produced by the
pro-reparative recruited macrophages and as a result of
complement cleavage peptides binding to their corresponding
receptors on cardiomyocytes, is a possible explanation. However,
studies on cell proliferation, using BrdU and anti-PCNA
(Figures 6,7) did not provide conclusive proof for this
mechanism. This is since much less labeling of proliferating
cardiomyocytes than that expected for the repopulation of the
debrided regions (gray areas in Figures 5B,G) was observed by
both methods.
Another possibility is that the myocardial repair is
associated with recruitment and activation of cardiac-resident
stem/progenitor cells, or of external mesenchymal stem
cells. Stem cells may differentiate into cardiomyocytes in
response to “cues” provided by the myocardial extracellular
matrix (ECM) and microenvironment. Alternatively, such
cells may have a paracrine effect on cell proliferation
within the injured myocardium (62). Previous studies have
suggested the existence of such external stem cells (e.g.,
bone marrow or adipose tissue stem cells) based on their
ability to differentiate in vitro into cardiomyocytes when
incubated with cardiac ECM and cardiac extracts (62–
64). In addition, sponge implants in mice, which had α-gal
nanoparticles, were found to contain both macrophages and
colony forming cells characteristic to stem cells, within several
days post-implantation (65).
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Galili et al. α-Gal Nanoparticles Reduce Infarct Size
FIGURE 7 | Immunofluorescence staining (IF) of proliferating cells by anti-PCNA antibody on Day 9 post-MI and intramyocardial α-gal nanoparticles injection. (A)
Intestinal villi demonstrating multiple proliferating cells with stained nuclei at the base of the crypts. (B,C) IF staining of the LV by anti-PCNA antibody. Only small
number of stained nuclei is observed. (D) H&E staining of the LV presented in (B,C). Data from one mouse of 5 with similar results.
In conclusion, this study is the first to demonstrate a
profound reduction in myocardial infarct size, restoration of
normal LV function and prevention of extensive fibrosis by
intramyocardial injection of α-gal nanoparticles in adult mice
after reperfusion, post-MI. The mechanism(s) mediating this
repair await elucidation. Future studies include a confirmatory
large animal model trial. If these dramatic benefits of α-
gal nanoparticles treatment are reproduced in a large animal
model of infarction and reperfusion, a clinical trial in patients
with ST-elevation myocardial infarction is warranted. Following
reperfusion of the occluded coronary artery with a stent in
the cardiac catheterization laboratory, there is an opportunity
to inject α-gal nanoparticles into the reperfused myocardium
using a percutaneous catheter technique (66–68) with the goal of
reducing infarct size and scar formation, improving LV function,
and ultimately reducing the progression to heart failure and
premature cardiac death. The large animal model planned for
such study is the GT-KO pig which lacks α-gal epitope because
of disruption of the α1,3GT gene (GGTA1) (42,43) and since it
produces the natural anti-Gal antibody in titers similar to those
in humans, without the need of immunization (69–71).
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included
in the article/Supplementary Material, further inquiries can be
directed to the corresponding author.
ETHICS STATEMENT
The animal study was reviewed and approved by IACUC
Committee Rush Medical College.
AUTHOR CONTRIBUTIONS
UG and GS developed the method, evaluated the histological and
echocardiography data, and wrote the manuscript. UG produced
the nanoparticles. ZZ performed the surgical, injection work,
and assisted in echocardiography studies. JC performed the
echocardiography studies and histological work. JG performed
the flow cytometry studies and the treatment of mice for
simulation of immune parameters as in humans. All authors
contributed to the article and approved the submitted version.
FUNDING
This work was funded by the Alvin H. Baum Family Fund,
Chicago, IL.
ACKNOWLEDGMENTS
We thank cardiology and research fellows at Rush University
Medical Center for their technical support (Drs. Issam Atalla,
Ashvarya Mangla, Anshuman Das, Ali Mahmood, Marie-France
Poulin, and Fareed Moses S. Collado). We also thank Dr.
Shu Qian Liu (Northwestern University), Dr. Dengping Yin
(University of Chicago School of Medicine), and Mr. Ayman
Isbatan for their technical assistance and Dr. Alan L. Landay for
his support in this project.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fcvm.
2021.719160/full#supplementary-material
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Galili et al. α-Gal Nanoparticles Reduce Infarct Size
REFERENCES
1. Minino AM, Murphy SL, Xu J, Kochanek KD. Deaths: final data for 2008. Natl
Vital Stat Rep. (2011) 59:1–126.
2. Shinde AV, Frangogiannis NG. Fibroblasts in myocardial infarction: a
role in inflammation and repair. J Mol Cell Cardiol. (2014) 70:74–
82. doi: 10.1016/j.yjmcc.2013.11.015
3. Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med. (1999) 341:738–
46. doi: 10.1056/NEJM199909023411006
4. Nahrendorf M, Swirski FK, Aikawa E, Stangenberg L, Wurdinger T,
Figueiredo JS, et al. The healing myocardium sequentially mobilizes two
monocyte subsets with divergent and complementary functions. J Exp Med.
(2007) 204:3037–47. doi: 10.1084/jem.20070885
5. Frantz S, Bauersachs J, Ertl G. Post-infarct remodeling: contribution
of wound healing and inflammation. Cardiovasc Res. (2009) 81:474–
81. doi: 10.1093/cvr/cvn292
6. Lavine KJ, Pinto AR, Epelman S, Kopecky BJ, Clemente-Casares X, Godwin
J, et al. The macrophage in cardiac homeostasis and disease: JACC
macrophage in CVD series (Part 4). J Am Coll Cardiol. (2018) 72:2213–
30. doi: 10.1016/j.jacc.2018.08.2149
7. Zhang S, Bories G, Lantz C, Emmons R, Becker A, Liu E, et al.
Immunometabolism of phagocytes and relationships to cardiac repair. Front
Cardiovasc Med. (2019) 6:42. doi: 10.3389/fcvm.2019.00042
8. Ferrini A, Stevens MM, Sattler S, Rosenthal N. Toward Regeneration of the
Heart: Bioengineering Strategies for Immunomodulation. Front Cardiovasc
Med. (2019) 6:26. doi: 10.3389/fcvm.2019.00026
9. Poss KD, Wilson LG, Keating MT. Heart regeneration in zebrafish. Science.
(2002) 298:2188–90. doi: 10.1126/science.1077857
10. Becker RO, Chapin S, Sherry R. Regeneration of the ventricular myocardium
in amphibians. Nature. (1974) 248:145–7. doi: 10.1038/248145a0
11. Flink IL. Cell cycle reentry of ventricular and atrial cardiomyocytes and cells
within the epicardium following amputation of the ventricular apex in the
axolotl, Amblystoma mexicanum: confocal microscopic immunofluorescent
image analysis of bromodeoxyuridine-labeled nuclei. Anat Embryol (Berl).
(2002) 205:235–44. doi: 10.1007/s00429-002-0249-6
12. Porrello ER, Mahmoud AI, Simpson E, Hill JA, Richardson JA, Olson EN, et al.
Transient regenerative potential of the neonatal mouse heart. Science. (2011)
331:1078–80. doi: 10.1126/science.1200708
13. Haubner BJ, Adamowicz-Brice M, Khadayate S, Tiefenthaler V,
Metzler B, Aitman T, et al. Complete cardiac regeneration in a
mouse model of myocardial infarction. Aging (Albany NY). (2012)
4:966–77. doi: 10.18632/aging.100526
14. Ye L, D’Agostino G, Loo SJ, Wang CX, Su LP, Tan SH, et al. Early
regenerative capacity in the porcine heart. Circulation. (2018) 138:2798–
808. doi: 10.1161/CIRCULATIONAHA.117.031542
15. Zhu W, Zhang E, Zhao M, Chong Z, Fan C, Tang Y, et al.
Regenerative potential of neonatal porcine hearts. Circulation. (2018)
138:2809–16. doi: 10.1161/CIRCULATIONAHA.118.034886
16. Evans MA, Smart N, Dubé KN, Bollini S, Clark JE, Evans HG, et al. Thymosin
β4-sulfoxide attenuates inflammatory cell infiltration and promotes cardiac
wound healing. Nat Commun. (2013) 4:2081. doi: 10.1038/ncomms3081
17. Godwin JW, Pinto AR, Rosenthal NA. Macrophages are required for adult
salamander limb regeneration. Proc Natl Acad Sci USA. (2013) 110:9415–
20. doi: 10.1073/pnas.1300290110
18. Aurora AB, Porrello ER, Tan W, Mahmoud AI, Hill JA, Bassel-Duby R,
et al. Macrophages are required for neonatal heart regeneration. J Clin Invest.
(2014) 124:1382–92. doi: 10.1172/JCI72181
19. Rubin N, Harrison MR, Krainock M, Kim R, Lien CL. Recent
advancements in understanding endogenous heart regeneration-insights
from adult zebrafish and neonatal mice. Semin Cell Dev Biol. (2016)
58:34–40. doi: 10.1016/j.semcdb.2016.04.011
20. Bolaños-Castro LA, Walters HE, García Vázquez RO, Yun MH. Immunity in
salamander regeneration: Where are we standing and where are we headed?
Dev Dyn. (2021) 250:753–67. doi: 10.1002/dvdy.251
21. Del Rio-Tsonis K, Tsonis PA, Zarkadis IK, Tsagas AG, Lambris
JD. Expression of the third component of complement, C3, in
regenerating limb blastema cells of urodeles. J Immunol. (1998)
161:6819–24. doi: 10.1016/S0161-5890(98)90697-6
22. Mastellos DC, Deangelis RA, Lambris JD. Complement-triggered pathways
orchestrate regenerative responses throughout phylogenesis. Semin Immunol.
(2013) 25:29–38. doi: 10.1016/j.smim.2013.04.002
23. Natarajan N, Abbas Y, Bryant DM, Gonzalez-Rosa JM, Sharpe M, Uygur
A, et al. Complement receptor C5aR1 plays an evolutionarily conserved
role in successful cardiac regeneration. Circulation. (2018) 137:2152–
65. doi: 10.1161/CIRCULATIONAHA.117.030801
24. Galili U, Wigglesworth K, Abdel-Motal UM. Accelerated healing of skin
burns by anti-Gal/α-gal liposomes interaction. BURNS. (2010) 36:239–
51. doi: 10.1016/j.burns.2009.04.002
25. Wigglesworth KM, Racki WJ, Mishra R, Szomolanyi-Tsuda E, Greiner DL,
Galili U. Rapid recruitment and activation of macrophages by anti-Gal/α-gal
liposome interaction accelerates wound healing. J Immunol. (2011) 186:4422–
32. doi: 10.4049/jimmunol.1002324
26. Hurwitz Z, Ignotz R, Lalikos J, Galili U. Accelerated porcine wound
healing with α-gal nanoparticles. Plas Reconstruc Surg. (2012) 129:242–
51. doi: 10.1097/PRS.0b013e31823aebb1
27. Galili U. α-Gal nanoparticles in wound and burn healing acceleration. Adv
Wound Care. (2017) 6:81–91. doi: 10.1089/wound.2016.0703
28. Kaymakcalan OE, Karinja S, Abadeer A, Dong X, Jin JL, Galili U,
et al. Antigen-mediated, macrophage-stimulated, accelerated wound healing
using α-gal nanoparticles. Ann Plast Surg. (2018) 80(Suppl. 4):S196–
203. doi: 10.1097/SAP.0000000000001360
29. Kaymakcalan OE, Abadeer A, Goldufsky JW, Galili U, Karinja SJ, Dong X,
et al. Topical α-gal nanoparticles accelerate diabetic wound healing. Exp
Dermatol. (2020) 29:404–13. doi: 10.1111/exd.14084
30. Galili U. Anti-Gal: An abundant human natural antibody of
multiple pathogeneses and clinical benefits. Immunology. (2013)
140:1–11. doi: 10.1111/imm.12110
31. Galili U, Rachmilewitz EA, Peleg A, Flechner I. A unique natural human
IgG antibody with anti-α-galactosyl specificity. J Exp Med. (1984) 160:1519–
31. doi: 10.1084/jem.160.5.1519
32. Galili U, Macher BA, Buehler J, Shohet SB. Human natural anti-alpha-
galactosyl IgG. II. The specific recognition of alpha (1—-3)-linked galactose
residues. J Exp Med. (1985) 162:573–82. doi: 10.1084/jem.162.2.573
33. Towbin H, Rosenfelder G, Wieslander J, Avila JL, Rojas M, Szarfman
A, et al. Circulating antibodies to mouse laminin in Chagas disease,
American cutaneous leishmaniasis, and normal individuals recognize
terminal galactosyl(alpha 1-3)-galactose epitopes. J Exp Med. (1987) 166:419–
32. doi: 10.1084/jem.166.2.419
34. Galili U, Clark MR, Shohet SB, Buehler J, Macher BA. Evolutionary
relationship between the anti-Gal antibody and the Galα1-
3Gal epitope in primates. Proc Natl Acad Sci USA. (1987)
84:1369–73. doi: 10.1073/pnas.84.5.1369
35. Galili U, Shohet SB, Kobrin E, Stults CLM, Macher BA. Man, apes, and
Old-World monkeys differ from other mammals in the expression of α-
galactosyl epitopes on nucleated cells. J Biol Chem. (1988) 263:17755–
62. doi: 10.1016/S0021-9258(19)77900-9
36. Teranishi K, Mañez R, Awwad M, Cooper DK. Anti-Gal (1-3Gal IgM and
IgG antibody levels in sera of humans and Old World non-human primates.
Xenotransplantation. (2002) 9:148–54. doi: 10.1034/j.1399-3089.2002.1o058.x
37. Galili U, Swanson K. Gene sequences suggest inactivation of α-1,3-
galactosyltransferase in catarrhines after the divergence of apes from monkeys.
Proc Natl Acad Sci USA. (1991) 88:7401–4. doi: 10.1073/pnas.88.16.7401
38. Thall AD, Malý P, Lowe JB. Oocyte Galα1,3Gal epitopes implicated
in sperm adhesion to the zona pellucida glycoprotein ZP3 are not
required for fertilization in the mouse. J Biol Chem. (1995) 270:21437–
40. doi: 10.1074/jbc.270.37.21437
39. Tanemura M, Yin D, Chong AS, Galili U. Differential immune response to
α-gal epitopes on xenografts and allografts: Implications for accommodation
in xenotransplantation. J Clin Invest. (2000) 105:301–10. doi: 10.1172/JC
I7358
40. Galili U. Wigglesworth K, Abdel-Motal UM. Intratumoral injection of α-gal
glycolipids induces and conversion of lesions into endogenous vaccines. J
Immunol. (2007) 178:4676–87. doi: 10.4049/jimmunol.178.7.4676
41. Tanemura M, Maruyama S, Galili U. Differential expression of α-gal epitopes
(Galα1-3Galß1-4GlcNAc-R) on pig and mouse organs. Transplantation.
(2000) 69:187–90. doi: 10.1097/00007890-200001150-00034
Frontiers in Cardiovascular Medicine | www.frontiersin.org 13 August 2021 | Volume 8 | Article 719160

Galili et al. α-Gal Nanoparticles Reduce Infarct Size
42. Lai L, Kolber-Simonds D, Park KW, Greenstein JL, Im GS, Samuel M, et al.
Production of α-1,3-galactosyltransferase knockout pigs by nuclear transfer
cloning. Science. (2002) 295:1089–92. doi: 10.1126/science.1068228
43. Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen SH, et al.
Production of α1,3-galactosyltransferase-deficient pigs. Science. (2003)
299:411–4. doi: 10.1126/science.1078942
44. Yang F, Liu YH, Yang XP, Xu J, Kapke A, Carretero OA. Myocardial
infarction and cardiac remodelling in mice. Exp Physiol. (2002) 87:547–
55. doi: 10.1113/eph8702385
45. Vandervelde S, van Amerongen MJ, Tio RA, Petersen AJ, van Luyn MJA,
Harmsen MC. Increased inflammatory response and neovascularization in
reperfused vs. non-reperfused murine myocardial infarction. Cardio Vasc
Pathol. (2006) 15:83–90. doi: 10.1016/j.carpath.2005.10.006
46. Dong R, Liu P, Wee L, Butany J, Sole MJ. Verapamil ameliorates the clinical
and pathological course of murine myocarditis. J Clin Invest. (1992) 90:2022–
30. doi: 10.1172/JCI116082
47. De Celle T, Cleutjens JP, Blankesteijn WM, Debets JJ, Smits JF, Janssen
BJ. Long-term structural and functional consequences of cardiac
ischaemia-reperfusion injury in vivo in mice. Exp Physiol. (2004)
89:605–15. doi: 10.1113/expphysiol.2004.027649
48. Tsutsumi YM, Patel HH, Lai NC, Takahashi T, Head BP, Roth
DM. Isoflurane produces sustained cardiac protection after
ischemia-reperfusion injury in mice. Anesthesiology. (2006)
104:495–502. doi: 10.1097/00000542-200603000-00017
49. Ananthakrishnan R, Li Q, O’Shea KM, Quadri N, Wang L, Abuchowski A,
et al. Carbon monoxide form of PEGylated hemoglobin protects myocardium
against ischemia/reperfusion injury in diabetic and normal mice. Artif Cells
Nanomed Biotechnol. (2013) 41:428–36. doi: 10.3109/21691401.2012.762370
50. Dutta P, Nahrendorf M. Monocytes in myocardial infarction. Arterioscler
Thromb Vasc Biol. (2015) 35:1066–70. doi: 10.1161/ATVBAHA.114.304652
51. Shiraishi M, Shintani Y, Shintani Y, Ishida H, Saba R, Yamaguchi A, et al.
Alternatively activated macrophages determine repair of the infarcted adult
murine heart. J Clin Invest. (2016) 126:2151–66. doi: 10.1172/JCI85782
52. Christia P, Bujak M, Gonzalez-Quesada C, Chen W, Dobaczewski M, Reddy
A, et al. Systematic characterization of myocardial inflammation, repair,
and remodeling in a mouse model of reperfused myocardial infarction. J
Histochem Cytochem. (2013) 61:555–70. doi: 10.1369/0022155413493912
53. Castellan RFP, Meloni M. Mechanisms and therapeutic targets of
cardiac regeneration: closing the age gap. Front Cardiovasc Med. (2018)
5:7. doi: 10.3389/fcvm.2018.00007
54. Mahmoud AI, Porrello ER. Turning back the cardiac regenerative
clock: lessons from the neonate. Trends Cardiovasc Med. (2012) 22:128–
33. doi: 10.1016/j.tcm.2012.07.008
55. Frangogiannis NG. Cell biological mechanisms in regulation of the
post-infarction inflammatory response. Curr Opin Physiol. (2018) 1:7–
13. doi: 10.1016/j.cophys.2017.09.001
56. Psarras S, Beis D, Nikouli S, Tsikitis M, Capetanaki Y. Three in a box:
understanding cardiomyocyte, fibroblast, and innate immune cell interactions
to orchestrate cardiac repair processes. Front Cardiovasc Med. (2019)
6:32. doi: 10.3389/fcvm.2019.00032
57. Nahrendorf M, Swirski FK, Abandoning M1/M2 for a network
model of macrophage function. Circ Res. (2016) 119:414–
7. doi: 10.1161/CIRCRESAHA.116.309194
58. Kumar AG, Ballantyne CM, Michael LH, Kukielka GL, Youker KA, Lindsey
ML, et al. Induction of monocyte chemoattractant protein-1 in the small
veins of the ischemic and reperfused canine myocardium. Circulation. (1997)
95:693–700. doi: 10.1161/01.CIR.95.3.693
59. Frangogiannis NG, Dewald O, Xia Y, Ren G, Haudek S, Leucker T, et al.
Critical role of monocyte chemoattractant protein-1/CC chemokine ligand 2
in the pathogenesis of ischemic cardiomyopathy. Circulation. (2007) 115:584–
92. doi: 10.1161/CIRCULATIONAHA.106.646091
60. Nimmerjahn F, Ravetch JV. Fcγreceptors: old friends and new family
members. Immunity. (2006) 24:19–28. doi: 10.1016/j.immuni.2005.11.010
61. Vogelpoel LT, Baeten DL, de Jong EC, den Dunnen J. Control of cytokine
production by human Fcγreceptors: implications for pathogen defense and
autoimmunity. Front Immunol. (2015) 6:79. doi: 10.3389/fimmu.2015.00079
62. Maghin E, Garbati P, Quarto R, Piccoli M, Bollini S. Young at heart: combining
strategies to rejuvenate endogenous mechanisms of cardiac repair. Front
Bioeng Biotechnol. (2020) 8:447. doi: 10.3389/fbioe.2020.00447
63. Perán M, Marchal JA, López E, Jiménez-Navarro M, Boulaiz H, Rodríguez-
Serrano F, et al. Human cardiac tissue induces transdifferentiation of
adult stem cells towards cardiomyocytes. Cytotherapy. (2010) 12:332–
7. doi: 10.3109/14653240903548202
64. Gaustad KG, Boquest AC, Anderson BE, Gerdes AM, Collas P.
Differentiation of human adipose tissue stem cells using extracts
of rat cardiomyocytes. Biochem Biophys Res Commun. (2004)
314:420–7. doi: 10.1016/j.bbrc.2003.12.109
65. Galili U. The Natural Anti-Gal Antibody as Foe Turned Friend in Medicine.
London: Academic Press/Elsevier. (2018). p. 245.
66. Patel AN, Henry TD, Quyyumi AA, Schaer GL, Anderson RD, Toma C, et al.
and ixCELL-DCM Investigators. Ixmyelocel-T for patients with ischaemic
heart failure: a prospective randomised double-blind trial. Lancet. (2016)
387:2412–21. doi: 10.1016/S0140-6736(16)30137-4
67. Povsic TJ, Henry TD, Traverse JH, Fortuin FD, Schaer GL, Kereiakes
DJ, et al. and RENEW Investigators. The RENEW Trial: Efficacy and
Safety of Intramyocardial Autologous CD34(+) Cell Administration in
Patients With Refractory Angina. JACC Cardiovasc Interv. (2016) 9:1576–
85. doi: 10.1016/j.jcin.2016.05.003
68. Kumar A, Haralampus CA, Hughes M, Rouy D, Cresswell N. Braun R,
et al. Assessment of safety, accuracy and human CD34+cell retention after
intramyocardial injections with a helical needle catheter in a porcine model.
Catheter Cardiovasc Interv. (2013) 81:970–7. doi: 10.1002/ccd.24476
69. Dor FJ, Tseng YL, Cheng J, Moran K, Sanderson TM, Lancos CJ,
et al. α1,3-Galactosyltransferase gene-knockout miniature swine produce
natural cytotoxic anti-Gal antibodies. Transplantation. (2004) 78:15–
20. doi: 10.1097/01.TP.0000130487.68051.EB
70. Fang J, Walters A, Hara H, Long C, Yeh P, Ayares D, et al. Anti-Gal antibodies
in α1,3-galactosyltransferase gene-knockout pigs. Xenotransplantation. (2012)
19:305–10. doi: 10.1111/j.1399-3089.2012.00710.x
71. Galili U. α1,3Galactosyltransferase knockout pigs produce the natural anti-
Gal antibody and simulate the evolutionary appearance of this antibody
in primates. Xenotransplantation. (2013) 20:267–76. doi: 10.1111/xen.
12051
Conflict of Interest: UG is the inventor of patent #8865178 “Compositions
and methods for wound healing” which includes the therapy described in this
manuscript. The patent is owned by the University of Massachusetts.
The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest.
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Frontiers in Cardiovascular Medicine | www.frontiersin.org 14 August 2021 | Volume 8 | Article 719160