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Integrative Physiology
Intravenously Delivered Mesenchymal Stem Cells
Systemic Anti-Inflammatory Effects Improve Left Ventricular Dysfunction
in Acute Myocardial Infarction and Ischemic Cardiomyopathy
Dror Luger,* Michael J. Lipinski,* Peter C. Westman, David K. Glover, Julien Dimastromatteo,
Juan C. Frias, M. Teresa Albelda, Sergey Sikora, Alex Kharazi, Grigory Vertelov,
Ron Waksman, Stephen E. Epstein
Rationale: Virtually all mesenchymal stem cell (MSC) studies assume that therapeutic effects accrue from local
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myocardial effects of engrafted MSCs. Because few intravenously administered MSCs engraft in the myocardium,
studies have mainly utilized direct myocardial delivery. We adopted a different paradigm.
Objective: To test whether intravenously administered MSCs reduce left ventricular (LV) dysfunction both post–
acute myocardial infarction and in ischemic cardiomyopathy and that these effects are caused, at least partly, by
systemic anti-inflammatory activities.
Methods and Results: Mice underwent 45 minutes of left anterior descending artery occlusion. Human
MSCs, grown chronically at 5% O2, were administered intravenously. LV function was assessed by serial
echocardiography, 2,3,5-triphenyltetrazolium chloride staining determined infarct size, and fluorescenceactivated cell sorting assessed cell composition. Fluorescent and radiolabeled MSCs (1×106) were injected 24 hours
post–myocardial infarction and homed to regions of myocardial injury; however, the myocardium contained only
a small proportion of total MSCs. Mice received 2×106 MSCs or saline intravenously 24 hours post–myocardial
infarction (n=16 per group). At day 21, we harvested blood and spleens for fluorescence-activated cell sorting and
hearts for 2,3,5-triphenyltetrazolium chloride staining. Adverse LV remodeling and deteriorating LV ejection
fraction occurred in control mice with large infarcts (≥25% LV). Intravenous MSCs eliminated the progressive
deterioration in LV end-diastolic volume and LV end-systolic volume. MSCs significantly decreased natural killer
cells in the heart and spleen and neutrophils in the heart. Specific natural killer cell depletion 24 hours pre–
acute myocardial infarction significantly improved infarct size, LV ejection fraction, and adverse LV remodeling,
changes associated with decreased neutrophils in the heart. In an ischemic cardiomyopathy model, mice 4 weeks
post–myocardial infarction were randomized to tail-vein injection of 2×106 MSCs, with injection repeated at week
3 (n=16) versus PBS control (n=16). MSCs significantly increased LV ejection fraction and decreased LV endsystolic volume.
Conclusions: Intravenously administered MSCs for acute myocardial infarction attenuate the progressive
deterioration in LV function and adverse remodeling in mice with large infarcts, and in ischemic cardiomyopathy,
they improve LV function, effects apparently modulated in part by systemic anti-inflammatory activities. (Circ Res. 2017;120:1598-1613. DOI: 10.1161/CIRCRESAHA.117.310599.)
Key Words: cardiomyopathies
■
inflammation
■
killer cells, natural
A
lthough percutaneous coronary intervention has improved
outcomes for patients with acute myocardial infarction
■
myocardial infarction
■
stem cells
(AMI), some patients with AMI, including those with minimal
evidence of left ventricular (LV) dysfunction, develop progressive adverse LV remodeling despite optimal medical therapy.1
Importantly, after AMI, the immune response, particularly the
innate response, plays a crucial role in both myocardial recovery and remodeling processes.2 We and others suggested that
Editorial, see p 1530
In This Issue, see p 1519
Meet the First Author, see p 1520
Original received January 8, 2017; revision received February 17, 2017; accepted February 23, 2017. In January 2017, the average time from submission
to first decision for all original research papers submitted to Circulation Research was 13.77 days.
From the MedStar Washington Hospital Center, Washington, DC (D.L., M.J.L., P.C.W., R.W., S.E.E.); University of Virginia, Charlottesville (D.K.G.,
J.D.); Universidad CEU Cardenal Herrera, Valencia, Spain (J.C.F.); GIBI230, Grupo de Investigación Biomédica en Imagen, IIS La Fe, Valencia, Spain
(M.T.A.); and CardioCell Inc, San Diego, CA (S.S., A.K., G.V.).
*These authors contributed equally to this article.
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.
117.310599/-/DC1.
Correspondence to Stephen E. Epstein, MD, MedStar Heart and Vascular Institute, MedStar Washington Hospital Center, 110 Irving St, NW, Washington,
DC 20010. E-mail [email protected]
© 2017 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.117.310599
1598
Luger et al Intravenously Delivered Mesenchymal Stem Cells 1599
Novelty and Significance
What Is Known?
• Stem cell therapeutic efficacy has been thought to depend on local
myocardial effects, necessitating efficient myocardial engraftment.
• Intravenous delivery of stem cells results in low cardiac engraftment
leading to a focus on catheter-based delivery.
• Continuing inflammation probably contributes to progressive myocardial dysfunction occurring in patients after a myocardial infarction and
in patients with ischemic or nonischemic cardiomyopathy.
What New Information Does This Article Contribute?
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• Intravenous administered mesenchymal stem cells (MSCs) inhibit progressive myocardial deterioration that occurs after myocardial infarction and during chronic heart failure.
• The improved outcomes of cell therapy seem to be related to potent
systemic anti-inflammatory effects.
• Stem cells do not have to be delivered directly to the heart to improve
cardiac function.
It is generally assumed that for stem cells to improve myocardial
function in patients, large numbers of cells need to engraft in
Nonstandard Abbreviations and Acronyms
AMI
LV
LVEDV
LVEF
LVESV
MI
MSC
NK
acute myocardial infarction
left ventricular
left ventricular end-diastolic volume
left ventricular ejection fraction
left ventricular end-systolic volume
myocardial infarction
mesenchymal stem cell
natural killer
these same proinflammatory immune responses, essential for
myocardial healing, may be excessive and thereby constitute a
major cause of progressive adverse remodeling and myocardial
dysfunction.1,3–5 A similar evolution has occurred in our concepts relating to the mechanisms responsible for progressive
LV dysfunction in patients with chronic ischemic or nonischemic cardiomyopathy. Thus, excessive immune/inflammatory
responses may importantly contribute to these patients’ progressive myocardial dysfunction.
This inflammatory concept of disease progression has led
us to explore the potential of a particular strategy that has the
capacity not only to modulate inflammatory responses but
also to favorably alter other mechanisms thought to contribute
to progressive LV dysfunction. That strategy is the systemic
administration of mesenchymal stem cells (MSCs).
Our focus on MSCs derives from coupling the inflammatory
hypothesis of progressive cardiac dysfunction post-AMI and in
patients with cardiomyopathy, with known activities of MSCs.
Among these are their immunomodulatory effects, leading to
suppression of both innate and adaptive immune responses.6,7
In addition, the ability of MSCs to secrete a broad array of factors8 influencing, for example, adverse LV remodeling, angiogenesis, tissue healing, apoptosis, mitochondrial dysfunction,
the myocardium to exert local effects. Because intravenous delivery of MSCs leads to sparse myocardial engraftment, focus has
been almost exclusively on catheter delivery of cells, an impractical strategy for repeated administrations. Chronic inflammation
seems to contribute to progressive myocardial deterioration that
occurs after infarction and in patients with heart failure. Because
MSCs secrete factors with multiple activities, including antiinflammatory effects, we postulated that intravenously administered MSCs could improve clinical outcomes, in part, by systemic
anti-inflammatory effects. MSCs were injected intravenously into
mice with myocardial infarction or with ischemic cardiomyopathy. Marked improvements in left ventricular function occurred,
which were associated with anti-inflammatory effects. An MSCinduced decrease in NK cells seemed to play an important role
in their beneficial cardiac effects. This study shows that despite
low myocardial engraftment, intravenously administered MSCs
improve cardiac function in both acute myocardial infarction and
ischemic cardiomyopathy, effects modulated in part by systemic
anti-inflammatory effects.
microvascular dysfunction, and collagen deposition,9–14 raises
the possibility that their multitarget functionality could, separately and synergistically, increase the likelihood of this strategy leading to clinically important inhibition of adverse LV
remodeling and progressive deterioration of LV function.
Another consideration of major practical consequence is the
concept, driving the design of most preclinical and clinical studies, that any beneficial effect of MSCs derives from their local
effects. It is commonly thought that once engrafted in damaged
myocardium, stem cells either directly transdifferentiate into
functional myocardium, stimulate resident myocardial stem
cells to expand and repopulate the heart with functioning myocytes, or secrete substances leading to myocardial repair.15–17
This orientation, along with the demonstration that stem cells,
administered intravenously, only poorly engraft in ischemic
myocardium, led to reliance on intracoronary, transendocardial,
or intramyocardial administration of stem cells.18–22
Our strategy for stem cell administration was based on a very
different paradigm. If an excessive and prolonged inflammatory
process is a key player in causing progressive cardiac dysfunction, then it is possible that MSCs, administered intravenously,
might exert through paracrine mechanisms systemic anti-inflammatory effects. Intravenous delivery would have an additional
advantage considering that the chronic processes leading to progressive LV dysfunction will probably never be cured by a single
administration of stem cells. If true, then repeated injections over
time would be necessary for a sustained therapeutic effect—a
situation in which the intravenous route of administration would
have a huge advantage over catheter-based delivery strategies.
We tested these concepts in studies on murine AMI and
ischemic cardiomyopathy models. We used standard ischemia/
reperfusion models of AMI (to simulate the clinical condition
that would exist in patients undergoing urgent percutaneous
coronary intervention for AMI) and of ischemic cardiomyopathy. MSCs were obtained from a young healthy human donor.
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We expanded the MSCs under chronic hypoxic conditions,
similar to their natural niche in the bone marrow, as exposure of
MSCs to these growth conditions enhances their functionality.23
Of relevance, hypoxia-preconditioning of cardiac progenitor
cells seems to improve cardiac outcome in experimental AMI.24
Thus, this study explores the efficacy of intravenously administered human MSCs, grown under chronic hypoxic conditions,
on (1) LV function after AMI, (2) LV function in ischemic cardiomyopathy, and (3) whether MSC-induced beneficial effects
derive, at least in part, from their immunomodulatory activities.
Methods
Ethics Statement
Animal handling and care followed the recommendations of the
National Institute of Health guide for care and use of laboratory animals. All protocols were approved by the animal care and use committee at Washington Hospital Center, Washington, DC.
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Animals
All animals used in the current study were male CD1 mice. Mice
were 8 to 10 weeks of age with body weight of 25 of 35 g.
AMI Model
AMI was produced by temporary ligation of the left anterior descending coronary artery for 45 minutes followed by reperfusion. Ligation
was considered successful when the anterior wall of the LV turned
pale (Online Methods). After AMI, mice were individually housed.
The top panel of Online Figure I depicts a schematic of the experimental protocol.
Human MSCs
Primary frozen bone marrow–derived human MSCs were received
from Stemedica (San Diego, CA). Culture conditions are described in
Online Methods. Unless otherwise specified, 2×106 MSCs in 0.2 mL
were injected in each mouse into the lateral tail vein 24 hours after
AMI (Online Methods).
Radiolabeled MSC Experiments
Cells were labeled with indium-111 oxine. Tail-vein injection of
1.0×106 radiolabeled MSCs was performed 24 hours after AMI
(n=10) and in control mice without myocardial infarction (MI; n=10;
Online Methods). Approximately 20 hours after intravenous injection
of radiolabeled MSCs, organs were harvested. Gamma well counting
and phosphor imaging (Perkin Elmer) was performed to assess radioactivity levels within the organs. Data were analyzed in a blinded
fashion (Sante DICOM software) with adjustment for the injected
radioactive dose per animal and background.
Ex Vivo Fluorescent Imaging
MSCs were labeled fluorescently with Qdot 705 (Invitrogen) or
ICG 820 (Sigma) and injected as above. To identify tissue-engrafted
MSCs, mice were euthanized and organs were harvested and analyzed using the In Vivo Imaging System (IVIS) 100 (Perkin Elmer)
with the relevant channel.
Ischemic Cardiomyopathy Model
Four weeks after AMI, mice were randomized based on their LV ejection fraction (EF) to either the MSC therapy group (n=16) or PBS
vehicle control injection group (n=16). Thus, mice in each group
will have comparable baseline LVEF before treatment. Mice then
underwent tail-vein injection and received either 2×106 MSCs in 0.2
mL of PBS or 0.2 mL of PBS control. After serial echocardiography
at weeks 1 and 3, mice received a repeat treatment after the week
3 echocardiogram. After echocardiography at weeks 4 and 6, mice
were euthanized after week 6 echocardiography and blood, heart,
and spleen were harvested for analysis. The bottom panel of Online
Figure I depicts a schematic of the experimental protocol.
Echocardiograms
Echocardiography (VEVO 1100 Imaging system; VisualSonics Inc)
was performed under light anesthesia. Short-axis images were acquired in a blinded fashion with corresponding M-mode imaging.
M-mode images were utilized to calculate LV functional parameters
(Online Methods).
Flow Cytometry
Single-cell suspensions for flow cytometry were prepared. Cells surface Fc receptors were blocked, and cells were stained with the relevant antibodies and viability marker. Flow cytometry was performed
on fluorescence-activated cell sorting Caliper (Becton Dickinson,
CA), and analysis was performed with FlowJo software (Ashland,
OR; Online Methods)
2,3,5-Triphenyltetrazolium Chloride staining.
After the day 20 echocardiograph, mice were euthanized and perfused by LV puncture using PBS. Short-axis cuts of the heart at 1-mm
thickness were incubated at 37°C in 1% 2,3,5-triphenyltetrazolium
chloride for 10 minutes. Heart sections were fixed in 2% paraformaldehide and photographed, and infarcted area was analyzed using
Image J software (NIH, Bethesda, MD; Online Methods).
In Vivo Natural Killer Cells Depletion
For in vivo natural killer (NK) cells depletion, the antibody antiNK1.1 clone was injected intravenously (100 mg/mouse) 24 hours
before MI surgery (n=12). Echocardiography was performed 7 and
21 days post-MI. At 21 days post-MI, infarct size (2,3,5-triphenyltetrazolium chloride) was analyzed and the ratio of spleen immune cells
was analyzed by fluorescence-activated cell sorting. Additional mice
were randomized to NK depletion or control and underwent AMI to
determine the impact of NK depletion on the immune cell content
within the spleen, heart, and blood 24 hours and 7 days after AMI
(Online Methods).
Results
Distribution of MSCs Administered IV
To determine in vivo distribution, human MSCs (1×106 per
mouse; n=12), radioactively or fluorescently labeled, were
administered intravenously to mice 24 hours after AMI or to
control mice without AMI.
Radiolabeled human MSCs were detected in all measured
organs at 24 hours post-injection (Figure 1A), showing high
concentrations in the liver, spleen, and kidneys. A small portion of MSCs engrafted in the heart. There was preferential engraftment into the heart after AMI compared with hearts from
animals without AMI (Figure 1B). Within MI hearts, MSCs
concentrated predominantly in regions of injury (Figures 1D
and 1E). Increased MI size strongly correlated with enhanced
MSC engraftment in the left anterior descending territory
(Figure 1F). Significantly higher MSC accumulation occurred
in spleens of MI mice (Figure 1C), indicating MI generated a
systemic signal, leading the spleen to express homing signals
for MSC accumulation.
This experiment was also performed with radiolabeled
murine MSCs (grown under chronic hypoxia); we found
greater murine MSC trafficking to the heart and spleen in
mice after AMI compared with mice the underwent sham surgery (Online Figure II). The data displayed in this figure also
provide comparative data between in vivo distribution of intravenously administered human and murine MSCs 24 hours
post-injection. There were significantly fewer human MSCs
that trafficked to the heart and spleen compared with murine
MSCs.
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Figure 1. In vivo biodistribution of mesenchymal stem cells (MSCs). Radiolabeled human MSCs were injected intravenously in mice
24 h post–myocardial infarction (MI) or in mice without MI (n=10 per group). MSC content in different organs was measured 24 h postinjection using a gamma well counter (A). Heart and spleen MSC engraftment was calculated per gram tissue (B and C). Percentage of
signal intensity in the left anterior descending (LAD) territory was determined from phosphor imaging of short-axis heart sections (D).
Representative phosphor images and 2,3,5-triphenyltetrazolium chloride (TTC) staining (to assess infarct size) of axial short-axis cross
sections of the heart are shown (E). Correlation between infarct size and MSC trafficking to the heart assessed by phosphor images is
presented (F). Analysis of MSC distribution on day 7 was accomplished by intravenous injection of fluorescently labeled MSCs 24 h postMI. Fluorescent intensity in heart sections was measured using an In Vivo Imaging System. G, A representative image is shown. The ratio
of MSCs engrafted into the heart by day 7 was analyzed by fluorescence-activated cell sorting on single-cell suspensions prepared from
whole heart. Viable MSCs were identified by staining with 2 human MSCs markers (CD90 and CD73),while gating on live cells (7AADnegative cells) (H). I, Heart engrafted MSCs were calculated as percentage of total viable heart cells.
The decreased engraftment of human versus murine
MSCs might be explained by our in vitro studies demonstrating complement-mediated lysis of human MSCs by
murine serum (data not shown). Despite this immune response to human MSCs, we demonstrated persistent viability
of human MSCs in the heart for as long as 3 weeks. Thus,
fluorescently labeled MSCs injected intravenously 24 hours
post-MI localized to the left anterior descending territory of
the heart and were seen on fluorescent imaging 7 days postMI (Figure 1G). Fluorescently labeled MSCs injected intravenously into mice with ischemic cardiomyopathy also localized
to the left anterior descending territory as seen on fluorescent
imaging 24 hours post-injection (Figure 1G). When gating on
live cells (7AAD-negative cells), flow cytometry of single-cell
1602 Circulation Research May 12, 2017
suspensions from the hearts demonstrated that human MSCs
within the myocardium were viable 7 days after injection in
the acute MI model (Figure 1H and 1I) and 3 weeks after injection in the ischemic cardiomyopathy model (see below;
Figure 4A).
Effects of MSCs on LVEF and LV Volumes
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Acute Myocardial Infarction
At day 21 post-MI, infarct size in the treated and control
groups did not significantly differ (Figure 2A). Although
LVEF, LV end-diastolic volume (EDV), and LV end-systolic
volume (ESV) significantly worsened over 3 weeks, the degree of deterioration was attenuated in the MSC-treated group;
however, the differences between the treated and control
groups were not statistically significant (Figure 2B through
2D). In controls, the end-diastolic and end-systolic wall thicknesses of both the anterior and posterior walls thinned over
the 3 weeks after AMI, changes compatible with progressive
LV dilatation unaccompanied by compensatory LV hypertrophy (Online Figure III). In contrast, MSC treatment prevented
significant end-systolic thinning of the both the anterior and
posterior walls (Online Figure III) and prevented significant
end-diastolic thinning of the posterior wall (Online Figure
III). Importantly, in addition to preventing the deleterious
changes occurring over time in the control group, when data
in the treated and control groups were directly compared, the
differences between the treated and control groups were statistically significant (Online Figure III).
Comparative Analysis of Mice With Small Versus Large
Infarcts
Further analysis of cardiac function revealed that MSC treatment improved adverse LV remodeling, but these effects were
limited to mice with MIs ≥25% of the LV (median infarct size
was 25%). In this subgroup, MSC treatment prevented the statistically significant increases in end-diastolic and end-systolic
volumes exhibited by the control group and there was a trend in
preventing a decrease in LVEF (Figure 2E through 2G).
Ischemic Cardiomyopathy
One mouse in the MSC treatment group with a severely reduced LVEF died when undergoing echocardiography and
tail-vein injection at week 3. As might be expected, 2 intravenous injections of MSCs in mice with ischemic cardiomyopathy did not reduce MI size compared with hearts from
PBS-injected control mice (29.6±9.1% versus 26.7±9.1%,
respectively; P=0.38; Figure 2H). In our model of ischemic
cardiomyopathy, baseline values represent echocardiographic
parameters 4 weeks post-MI. MSC treatment significantly increased LVEF from baseline, whereas there was no change in
LVEF in controls (Figure 2I). Interestingly, MSC treatment
significantly increased anterior and posterior wall thickness in
systole compared with control (Online Figure III). Although
the decrease in LVEDV with MSC treatment did not persist
(Figure 2J), MSC treatment significantly reduced LVESV
compared with control (Figure 2K). Thus, MSC treatment
led to persistent improvements in LV contraction compared
with control, reflected by significantly increased systolic wall
thickness, reduced LVESV, and increased LVEF.
Immunomodulatory Effects of MSCs
We found higher engraftment of MSCs in spleens of MI versus no MI mice (Figure 1C), suggesting AMI leads to a systemic signal that recruits MSCs to the spleen. We suspected
that this signal could also impact the spleen’s inflammatory
milieu. Flow cytometric analysis of the spleen 21 days after
AMI demonstrated that MSCs significantly reduced the percentage of splenic NK cells (Figure 3A and 3B), but did not
alter other cell subtypes in either lymphocytic or myeloid
compartments (Figure 3C).
To better understand the effect of MSCs on NK cells, we
explored the impact of MSCs injected 24 hours post-MI on the
composition of the different immune cells in the spleen at 7
days post-MI. Splenic NK cells were significantly increased in
mice 7 days post-MI (Figure 3D). Similar to the 21 days postMI study (Figure 3A and 3B), MSCs significantly reduced the
ratio of splenic NK cells when administered 24 hours post-MI
(Figure 3D) and did not alter the ratio of other immune cells
from both the lymphocytic and myeloid compartments (data
not shown). An MSC-induced decrease in NK cells was also
observed in the heart (Figure 3E). To verify that the reduction
in splenic NK cells is specific to the immunomodulatory effects of human MSCs and not related to a xenograft immune
response, we performed a separate experiment in which human
fibroblasts were injected 24 hours post-MI. When compared
with control mice that underwent MI, mice injected with human fibroblast 24 hours post-MI did not experience a decrease
in NK cells either in the spleen or the heart (Figure 3F).
Mechanism By Which MSCs Influence NK Cell Population
MSCs could affect NK cells through either direct cell to cell
contact or secreted factors. To determine whether MSCs impact NK cells via secreted factors, we conducted an in vitro
experiment using a transwell system (0.4-mm pore insert).
Murine splenocytes were placed in the insert and human MSCs
were placed in the wells in a gradient of 103 to 100×103 cells
per well, allowing cells to incubate for 96 hours. NK suppression by MSCs showed a dose–response effect (Figure 3G).
Thus, one of the mechanisms by which MSCs suppress NK
cells occurs via secreted factors.
Neutrophils are known as immediate responders to injury and have been shown to be major contributors to adverse
myocardial remodeling after MI.25,26 NK cells and neutrophils
have previously been shown to regulate each other.27 In light
of the ability of NK cells to regulate neutrophils, we explored
whether MSC-induced NK reduction has an effect on neutrophil content in the heart. Although there was not a significant
reduction of splenic neutrophils at 7 days post-MI after intravenous MSC administration (Figure 3C), we found that treatment with MSCs significantly reduced the accumulation of
neutrophils in the myocardium (Figure 3H and 3I). These data
suggest that human MSCs may either directly reduce myocardial NK cell and neutrophil content or the reduction in NK
cells could serve to reduce neutrophil infiltration, thus modulating the inflammatory process after AMI.
In the ischemic cardiomyopathy model, viable human
MSCs were present in the apex of hearts from the MSC treatment group (Figure 4A), demonstrating human MSCs can remain viable for 3 weeks after injection and evade the immune
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Figure 2. Intravenously administered human mesenchymal stem cell (MSCs) improve cardiac function in murine acute myocardial
infarction (MI) and ischemic cardiomyopathy models. MSCs were administered 24 h post-MI, and their therapeutic effect was
evaluated. Infarct size was calculated from 2,3,5-triphenyltetrazolium chloride (TTC)–stained heart sections (A). Echocardiography
was performed at baseline before MI and then again at days 3, 7, and 20 post-MI. Comparison is provided for left ventricular ejection
fraction (LVEF; B), LV end-diastolic volume (EDV; C), and LV end-systolic volume (ESV; D) for the MSC treatment vs control (Continued )
1604 Circulation Research May 12, 2017
Figure 2 Continued. groups. We also assessed the impact of MSCs on LV function and dimensions in mice with either small or large
infarct size (25% of LV cutoff), comparing day 20 LV functional data in hearts with small vs large infarcts in the same treatment group
(E–G). In the ischemic cardiomyopathy model (4 wk post-MI), mice received either intravenous MSC treatment or saline at baseline and
week 3 (n=16 per group). Serial echocardiography was performed at baseline, week 1, week 3, week 4, and week 6. Infarct size as a
percentage of the LV was determined by TTC staining of heart sections (H) to compare infarct size between the MSC treatment group
(MSCs) and PBS control group (no MSCs). Comparison is provided for LVEF (I), LVEDV (J), and LVESV (K) for the MSC treatment vs
control groups in the ischemic cardiomyopathy model. *P≤0.05, **P≤0.01 in comparison to baseline.
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response. To assess the impact of intravenously administered
human MSCs on splenic inflammatory cell content in the
ischemic cardiomyopathy model, the spleens of mice were
harvested and flow cytometry was performed. Comparison
of different immune cell composition in the spleen between mice that received MSC treatment and PBS control
is presented in the Table. Flow cytometry demonstrated that
intravenously administered MSCs significantly reduced
neutrophils (2.6±0.7 versus 4.9±1.3 for control; P<0.0001;
Figure 4B and 4C). However, there were no significant differences in the percentage of monocytes (Figure 4B and 4D).
Intravenous injection of MSCs significantly reduced myeloid
cells compared with control (18.5±2.5% versus 24.1±4.1%,
respectively, P<0.0001). There was also a trend toward reduction in NK cells with MSC treatment compared with control
(Table; Figure 4E). With the reduction in myeloid cells, there
was a resultant trend toward increased lymphocyte percentages with MSC treatment compared with control (77.9±11.0%
versus 71.8±12.3%, respectively; P=0.16). This resulted in a
significantly reduced splenic neutrophil:lymphocyte ratio for
MSC treatment compared with control (0.072±0.024 versus
0.146±0.057, respectively; P<0.0001).
We next sought to determine whether there are associations between the altered splenic cellular populations and LV
dimensions and function in the ischemic cardiomyopathy
model. Although MSC therapy significantly reduced the
levels of myeloid cell lines, there were no significant correlations between the percentage of splenic myeloid cells and
LV dimensions or function for either treated or control mice.
However, among mice that received MSC treatment, there
were significant associations between higher percentages of
splenic T lymphocytes, including CD3+, CD4+, and CD8+ T
lymphocytes, with improved LV remodeling and function:
that is, reduced LVESV, reduced LVEDV, and a greater LVEF
(Online Figure IV). These associations were not observed in
the control mice. Although splenic neutrophil:lymphocyte
ratio was not associated with infarct size (R2=0.00; P=0.92),
neutrophil:lymphocyte ratio strongly correlated with LVEF at
6 weeks (R2=0.31; P=0.001), a finding largely driven by the
immunomodulation associated with MSC treatment.
Causal Role of NK Cells in Deterioration of Cardiac
Function Post-AMI
We next sought to determine whether the reduction in NK cells
caused by intravenously administered MSCs plays a direct
causal role in the beneficial effects that MSCs exert on cardiac
function post-AMI. To answer this question, we determined
the effect of injecting intravenously, 24 hours before MI, an
antibody that depletes NK cells. Thus, we sought to determine
whether depletion of NK cells before AMI attenuated injury after AMI. In mice that underwent anti-NK1.1 antibodymediated NK cell depletion 24 hours before AMI, infarct
size 20 days post-MI was significantly smaller (Figure 5A).
Furthermore, NK cell depletion before AMI significantly attenuated the deterioration seen in cardiac function after AMI.
Importantly, LVEF was significantly higher and LVEDV and
LVESV were significantly lower in the NK depletion group
than in the control (Figure 5B through 5D).
NK Cells Support a Proinflammatory Immune
Response Post-MI
A single intravenous injection of anti-NK1.1 antibody 24
hours before MI maintained significantly lower splenic NK
cells at 20 days post-MI (Figure 5E and 5F). There was not
a significant effect on other immune cells in the lymphocytic
and myeloid compartments (Figure 5G). To determine how
NK cell depletion resulted in significant cardioprotection after AMI, we assess the impact of NK cell depletion on the
spleen and heart 24 hours and 7 days post-MI. NK cell depletion significantly reduced splenic neutrophil content 24 hours
post-MI, an effect that no longer remained significant 7 days
post-MI (Figure 6A and 6B). As expected with resolution of
the inflammatory response post-MI, there was a significant
reduction in splenic neutrophils at 7 days compared with 24
hours post-MI (Figure 6B). Interestingly, there was not a significant difference in splenic monocytes at either 24 hours
or 7 days post-MI (Figure 6A and 6C). Within the heart, NK
cell depletion significantly reduced neutrophil infiltration 24
hours post-MI (Figure 6D and 6E). However, there was no
longer a significant difference in myocardial neutrophils at 7
days post-MI. Interestingly, there was a significant increase in
myocardial monocytes at 24 hours and 7 days post-MI in the
NK depletion group (Figure 6F).
Discussion
Most previous stem cell studies have assumed that the underlying mechanism by which stem cells might exert beneficial
therapeutic effects in AMI or chronic cardiomyopathy derive
from local effects of stem cells that have engrafted in the injured myocardium.15–17 Because low numbers of stem cells
engraft in the myocardium after intravenous administration,
attention has focused, with rare exception,15–17,28 on refining
direct delivery of cells to the myocardium via catheter-based
delivery of cells.29–31
The current study design derives from several concepts.
First, that a persistent excessive inflammatory response exists
that probably contributes importantly to progressive myocardial dysfunction both in post-AMI and in cardiomyopathy
patients.3,4,32 Second, these chronic inflammatory responses
probably cannot be permanently suppressed by a single injection of a therapeutic. Thus, repeated injections will be necessary, a situation that would make catheter-based delivery
systems problematic. Third, MSCs secrete multiple factors
with a large array of activities, including anti-inflammatory
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Figure 3. Mesenchymal stem cells (MSCs) exert suppressive effects on natural killer (NK) cells and neutrophils. MSCs were
administered 24 h post-MI. The ratio of spleen NK cells was analyzed by fluorescence-activated cell sorting (FACS) at 21 d post-MI
(A and B) as well as other immune cells from lymphocyte and myeloid compartments (C; n=15 per group). The effect of MSCs on spleen
and heart NK cells was explored at day 7 post-MI by FACS analysis (D and E; n=8). Similarly, as a comparison to MSCs, the effect
of human fibroblasts on spleen and heart NK cells was tested by FACS analysis 7 d post-MI (F; n=7 per group). Effects of (Continued )
1606 Circulation Research May 12, 2017
Figure 3 Continued. MSC-secreted factors on spleen NK cells were tested in vitro in a transwell system. Spleen cells were placed in the
inserts that contained a mesh of 0.4-μm pores. The wells were populated with increasing numbers of MSCs (103 to 100×103). At the end
of 96-h incubation, NK cells ratio was analyzed by FACS (G). The impact of MSC treatment 24 h post-MI on cardiac neutrophils at day 7
post-MI was determined by FACS (H and I).
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effects.33–35 It therefore seems possible for beneficial cardiac
effects to accrue from not only MSCs engrafted locally in the
myocardium but also systemic anti-inflammatory actions of
products secreted by MSCs located in tissues other than the
myocardium.
This provided a major rationale for our testing the therapeutic efficacy of MSCs administered intravenously despite
low myocardial engraftment. We only found 2 previous publications, both preclinical, examining the effects of IV MSCs
on cardiac function and inflammation. Both demonstrated improved cardiac outcomes and, importantly, seemed to show
that MSCs exerted anti-inflammatory effects.28,36 Interpretation
of these 2 studies, however, is complicated by the fact that the
investigators infused human MSCs into immunocompromised
(NOD/SCID [nonobese diabetic/severe combined immunodeficiency]) mice, leaving uncertain what the immunomodulatory and salutary cardiac effects of the MSCs would be in an
immunocompetent animal.
Our initial experiments were designed to determine the
fate of human MSCs when administered intravenously 24
hours post-AMI. Similar to previous reports,36,37 we demonstrated that intravenously administered MSCs distributed to
multiple organs, but only a small portion homed to myocardium (Figure 1A)—these engrafted preferentially in ischemic
myocardium (Figure 1D and 1E). The myocardial-engrafted
cells persisted (Figure 1G) and were viable for at least 7 days
(Figure 1H and 1I). The number of engrafted cells directly related to the magnitude of LV infarct (Figure 1F). We also demonstrated that human MSCs injected intravenously into mice
with ischemic cardiomyopathy engrafted in the myocardium
(Figure 1G) and persisted in a viable state for at least 3 weeks
(Figure 4A). MSC numbers were also significantly higher in
spleens of mice with MI than those of naive mice (Figure 1C),
demonstrating myocardial injury generates systemic signals
that drive the cardiosplenic axis and recruit MSCs.
Effects of IV MSC Administration in Mice
With AMI
Our experiments demonstrated that infarct size was unchanged after MSC administration (Figure 2A). Furthermore,
we found that LV functional deterioration after AMI is not
a process completed after a few days. Rather, both adverse
LV remodeling (characterized by increasing LVEDV and
LVESV) and decreasing LVEF continue over the first few
weeks post-AMI (Figure 2B through 2D, control mice). There
was a trend suggesting that MSCs reduce this progressive
deterioration (Figure 2B through 2D). These positive trends
led us to examine the hypothesis that there might be greater
adverse LV remodeling in mice with large versus small infarcts, and if there was a benefit from MSC treatment, it would
likely be limited to the large infarct group. This speculation
proved correct. Treatment with MSCs prevented cardiac deterioration in mice with large infarcts, maintaining levels of
LVEF, LVEDV, and LVESV at values similar to those of mice
with small infarcts (Figure 2E through 2G). Thus, even though
most MSCs administered intravenously reside in tissues other
than the myocardium, intravenously administered MSCs prevent the adverse remodeling and deterioration in LV function
occurring >3 weeks after AMI in mice with large infarcts.
The immunomodulatory capacity of MSCs has been reported extensively.6,7 We sought to determine whether the
beneficial myocardial effects of MSCs are mediated, at least
partly, by modulating the inflammatory response triggered by
acute ischemic injury. We initially explored possible associations between MSC administration and changes in subpopulations of immune cells. Increasing data suggest the existence
of a functional cardiosplenic axis, in which the spleen alters
its immune/inflammatory cell populations in response to
signals produced by injured tissue, leading to mobilization
of immune/inflammatory cells that then home to the injured
tissue.38,39
At 7 days post-AMI, the percent of NK cells in the spleen
is significantly increased (Figure 3D). Treatment with MSCs
significantly reduced splenic NK cells to levels of naive mice
(Figure 3D). There was also a significant reduction in cardiac NK cells with MSCs (Figure 3E). We demonstrated that
this effect was not because of use of a xenograft model, as
administering human fibroblasts did not alter splenic or cardiac NK cell populations after AMI (Figure 3F). Figure 4E
demonstrates the serial changes observed in NK cell levels
present in the spleen over the time course encompassed by
the acute through the ischemic cardiomyopathy studies. NK
cells increase over the first week post-AMI and then gradually
decline. MSCs were able to maintain a lower NK cell level
throughout the period of the experiments.
Previous in vitro studies demonstrated that MSCimmunosuppressive effects on NK cells40,41 mediated, at
least partly, by secreted factors.42 In particular, IDO (indoleamine-pyrrole 2,3-dioxygenase) and PGE2 (prostaglandin
E2), secreted by MSCs, inhibited proliferation of NK cells
and suppressed their cytotoxic effects.42 PGE2 has also been
shown to increase apoptosis of NK cells.43,44 Although we did
not examine molecular mechanisms in our study, we found
that MSC-induced NK cell suppression is caused by soluble
factors, not requiring cell–cell contact (Figure 3G).
Importantly, MSCs administered intravenously 24 hours
post-MI significantly reduced cardiac neutrophils 7 days postAMI. (Figure 3H and 3I) The role of NK cells and neutrophils as mediators of inflammatory-induced cardiac functional
deterioration has been described.5,25,26,45 It also has been demonstrated that neutrophils and NK cells are tightly linked so
that either cell type can activate or regulate the other based on
pathophysiological conditions.27 Taking together the effect of
MSCs on NK cells and neutrophils, we suggest that a major
mechanism by which MSCs exert their beneficial myocardial
functional effects is by suppressing both NK cells and neutrophils (and thereby the proinflammatory interplay between
these cells).
Luger et al Intravenously Delivered Mesenchymal Stem Cells 1607
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Figure 4. Treatment with mesenchymal stem cells (MSCs) in cardiomyopathy (CM) suppresses neutrophils (Ly6CintGr1hi). In the
ischemic cardiomyopathy model (4 wk post-MI), mice received either intravenous MSC treatment or saline at baseline and week 3 (n=16
per group). At week 6, flow cytometry of the heart demonstrated viable human MSCs in the MSC treatment group (A). Spleen cells were
analyzed by fluorescence-activated cell sorting (B) to access the ratio of neutrophils (Ly6ChiGr1hi; C) and monocytes (Continued )
1608 Circulation Research May 12, 2017
Figure 4 Continued. (Ly6ChiGr1-; D). E, We provide a comparison plotting percent splenic natural killer (NK) cells (Y axis) in mice that
either received cell therapy or control over time since acute myocardial infarction (AMI; X-axis). The 10-week time point represents
ischemic cardiomyopathy mice that experience AMI and where randomized to cell therapy or control 4 wk after AMI. FSC indicates
forward scatter.
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To determine whether there was a causal relation between
the MSC-induced decrease in NK cells and the beneficial
effects of MSCs post-AMI, we performed NK1.1 antibodymediated NK cell depletion. NK cell depletion before AMI
onset decreased infarct size (Figure 5A). It also significantly
changed myocardial functional outcomes by day 20; NK depletion prevented LV adverse remodeling and improved LVEF
(Figure 5B through 5D). Yan et al46 also examined the effect of
NK cell depletion on LV function post-MI and reported no improvement in function. These investigators, however, limited
their study to 7 days, a time point at which we also showed
unaltered LV function.
Furthermore, we examined potential inflammationrelated mechanisms responsible for the functional effects
of NK cell depletion. We found that at 24 hours post-MI,
NK depletion significantly reduced the levels of neutrophils both in the spleen and in the heart (Figure 6A, 6B,
6D, and 6E). These results suggest that the immediate response of neutrophils to AMI is tightly linked to and regulated by NK cells. As neutrophil levels normally decrease
later in the course of AMI, as expected, we no longer found
a significant difference in splenic or cardiac neutrophil levels at 7 days post-MI despite NK depletion. Interestingly,
NK cell depletion significantly increased the proportion of
Ly6ChiGr1- monocytes at both 24 hours and 7 days post-MI
in the heart (Figure 6F), raising a question about the role of
these cells after AMI in the setting of reduced neutrophil
infiltration.38,47,48 It would therefore seem that NK cell depletion before AMI leads to an anti-inflammatory effect early
during AMI. Furthermore, the persistent NK depletion existing during the later healing phase of the AMI may further
contribute to the improved myocardial function we found in
the NK cell-depleted mice.
Table. Comparison of Splenic Cell Populations in the Ischemic
Cardiomyopathy Model in MSC-Treated and Control Mice
Splenic Cell Populations
No MSCs
MSCs
P Value
Ly6G Ly6C neutrophils, %
Ly6G− Ly6C+ monocytes, %
4.9±1.3
2.6±0.7
<0.0001
9.7±2.8
10.2±2.5
0.67
+
F4/80 Ly6C macrophages, %
4.2±2.8
3.7±2.9
0.68
NKp46 DX5 natural killer
cells, %
3.6±1.6
2.9±1.1
0.13
DX5+ CD3+ natural killer T
lymphocytes, %
1.6±0.5
1.8±0.7
0.58
CD3+ T lymphocytes, %
31.7±6.8
36.2±7.8
0.09
CD4 T lymphocytes, %
20.4±5.5
23.8±5.9
0.11
CD8 T lymphocytes, %
9.6±2.6
10.6±2.2
0.23
C19 B lymphocytes, %
39.3±8.6
42.6±7.4
0.26
+
+
+
+
+
+
+
+
Flow cytometry of splenocytes enabled comparison of the different immune
cell populations between the MSC treatment group and the PBS control group.
Populations are presented as a percentage of the total live splenocytes. Data are
presented as mean±SD.
Effects of IV MSC Administration in Mice With
Ischemic Cardiomyopathy
Given the impressive effects we found MSCs produce when
administered in the setting of AMI, we decided to examine,
in a randomized study, the same hypotheses we examined in
the AMI study that (1) intravenous administration of MSCs
in murine ischemic cardiomyopathy improves adverse LV remodeling and LV function and (2) one of the major mechanisms leading to such improvement is through a modulation
of immune/inflammatory pathways. Our results confirmed the
first hypothesis, and although we did not examine the second
hypothesis as rigorously as we did in the AMI model, our results were compatible with the validity of the second.
Mice that underwent acute MI (45 minutes of ischemia
followed by reperfusion) demonstrated clear echocardiographic evidence of cardiomyopathy after 4 weeks, exhibiting depressed LVEF along with elevated LVEDV and LVESV
(Figure 2I through 2K). MSCs were administered intravenously at 4 and at 7 weeks post-AMI.
MSC administration significantly increased LVEF by 27%
and decreased LVESV by 25%. These changes were associated with increased LV wall thickness in systole and reduced
LVESV. Meanwhile, the PBS control group experienced progressive adverse remodeling with a 3% reduction in LVEF
and a 9% increase in LVESV. MSC-induced improvements
became apparent as early as 1 to 3 weeks and, importantly,
persisted after the second administration of MSCs. There was
also a trend toward stabilization of LVEDV, with the MSC
treatment group demonstrating a 7% reduction in LVEDV,
whereas the PBS control group had an 8% increase in LVEDV
during the 6-week period.
Although fluorescent imaging (Figure 1G) and flow cytometry demonstrated that the intravenously administered
MSCs homed to the myocardium in these mice with ischemic
cardiomyopathy and remained viable (Figure 4A), only a
small percentage of the intravenously injected MSCs engraft
in the myocardium. Despite this, and similar to our studies in
acute MI, intravenously administered MSCs exerted systemic
immunomodulatory effects marked by a highly significant
decrease in splenic Ly6Cint/Ly6Ghi neutrophils (Figure 4B
and 4C). Although these proinflammatory cells have been
associated with adverse remodeling, they have a complex
role in MI and progression to heart failure.49 Recent studies
demonstrate that neutrophils play an integral role in switching macrophages to a reparative phenotype.47 This complex
interplay between resident macrophages and the innate immune response after MI50–53 requires further study, especially
given the impact MSCs play in modulating the inflammatory response after both acute MI and in chronic ischemic
cardiomyopathy.
MSCs also alter the balance existing between peripheral
and resident cardiac myeloid cell populations that orchestrate
repair after MI and modulate LV remodeling.54 As demonstrated in Online Figure IV, MSC treatment significantly modulates
Luger et al Intravenously Delivered Mesenchymal Stem Cells 1609
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Figure 5. In vivo natural killer (NK) cell depletion improves cardiac function post–myocardial infarction (MI). Antibody against NK1.1
was injected intravenously 24 h before MI induction (n=12 per group). Echocardiography was performed on days 7 and 20 post-MI. At 21
d post-MI, heart sections were stained with 2,3,5-triphenyltetrazolium chloride (TTC) and analyzed for infarct size, calculated as percent
of left ventricle (LV; A). Echocardiographic analysis included assessment of LV ejection fraction (B), LV end-diastolic volume (C), and LV
end-systolic volume (D). Fluorescence-activated cell sorting was performed on the spleen 21 d post-MI (n=12 per group), confirming NK
depletion (E and F) and assessed the composition of neutrophils and monocytes (G).
1610 Circulation Research May 12, 2017
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Figure 6. Natural killer (NK) cell depletion at the time of myocardial infarction (MI) in mice effects neutrophils (Ly6ChiGr1hi) and
monocytes (Ly6ChiGr1-) in the spleen and the heart. Anti-NK cell (NK1.1) antibody was administered intravenously immediately
after MI. The effect of NK depletion on the ratio of neutrophils (Ly6ChiGr1hi) and monocytes (Ly6ChiGr1-) was analyzed by fluorescenceactivated cell sorting, both at 24 h and at 7 days post-MI, in the spleen (A–C) and in the heart (D–F; n=8 in each group).
the T-lymphocyte populations, affecting the inflammatory
milieu and leading to a cardioprotective phenotype with reduced neutrophil:lymphocyte ratio, a finding supported in the
clinical literature.55 Our study therefore supports the concept
that MSC-mediated systemic immunomodulatory effects play
a role in the highly significant MSC-induced improvement
in LV function and in adverse LV remodeling in mice with
ischemic cardiomyopathy. Despite only a trend for reduction
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Luger et al Intravenously Delivered Mesenchymal Stem Cells 1611
in splenic NK cells with human MSCs in our ischemic cardiomyopathy model, a recent phase IIa randomized clinical
trial in nonischemic cardiomyopathy using these same human
MSCs demonstrated a significant reduction in circulating NK
cells. Interestingly, the degree of reduction significantly correlated with improvement in LVEF.56
Our findings contribute to the expanding body of data suggesting that the spleen plays an important role in mobilizing
the immune/inflammatory response to acute and chronic myocardial injury. The existence of a cardiosplenic axis was first
suggested by Swirski et al,38 who demonstrated rapid depletion of splenic monocytes shortly after acute MI, a change
accompanied by a massive influx of Ly-6Chigh monocytes
into ischemic myocardium; this influx was impaired by splenectomy. Ismahil et al39 demonstrated an increase in splenic
size in a murine model of chronic ischemic heart failure accompanied by a marked depletion of splenic proinflammatory
monocytes and an increased cardiac monocyte population.
Interestingly, splenectomy not only decreased cardiac macrophages and dendritic cells but also attenuated the adverse LV
remodeling. This supports an integral role for the spleen in
chronic heart failure. Although 18F-fluorodeoxyglucose positron emission tomographic imaging has validated the cardiosplenic axis in patients with acute MI,57 we are unaware of any
published studies examining the cardiosplenic axis in patients
with chronic heart failure.
Previous clinical trials targeting inflammation in heart
failure have failed to show clinical benefit.58,59 However, these
trials focused on inhibition of inflammation largely through
a single mechanism. MSCs secrete numerous growth factors
and cytokines influencing a diverse array of pathways, such
as those related to multiple inflammatory pathways, adverse
LV remodeling, angiogenesis, tissue healing, apoptosis, mitochondrial dysfunction, microvascular dysfunction, and collagen deposition.9–14 Such multifunctional activities provide
a rationale for why MSCs might provide a more effective
therapeutic strategy than drugs with a more limited range of
activities.
to exert a therapeutic effect. The literature supports a positive
response to this question.60–63 Most relevantly, our data indicate
that viable human MSCs persist for at least 20 days when administered IV into mice and that this duration of persistence is
sufficient to lead to significant immunomodulatory effects and
to marked improvement in LV function.
Issues Relating to the Xenograft Model and to the
Immune Status of MSCs
A critical question that must be addressed about the validity
of our results is whether the xenograft model we used, in
which human MSCs are injected into mice, provides results
that would be expected to be seen when allogenic human
MSCs are injected into humans. The literature on this subject is extensive60–62 and is discussed in detail in the Online
Discussion.
The bottom line is that MSCs are not immune privileged, as
originally proposed. They eventually are eliminated from mismatched hosts through immune responses. This probably explains, in part, the fact that fewer human versus murine MSCs
engrafted in the myocardium and spleen 24 hours after injection
in mice with AMI (Online Figure II). However, MSCs do persist
for a limited time (longer than other types of mismatched cells)
and are, thus, immune evasive but not immune privileged.60,62
Therefore, in either xenograft models of disease or in the setting
of administering allogenic MSCs to patients with cardiac (or
other) diseases, the issue is whether MSCs persist long enough
Conclusion
In an occlusion/reperfusion model of AMI, progressive adverse LV remodeling and deterioration in LV function occurs
during the first few weeks post-AMI in mice with large infarcts. Importantly, intravenously administered MSCs prevent
these deleterious effects, with a major contributory mechanism being an MSC-induced modulation of immune/inflammatory responses. This effect includes a suppressive effect
on NK cells, which we show decreases in both splenic and
cardiac neutrophils and improves adverse remodeling and
LV function. These results are compatible with the concept
that excessive inflammation is an important mechanism contributing to progressive LV dysfunction post-AMI, and that
at least one of the mechanisms responsible for the beneficial
myocardial effects of intravenously administered MSCs is
through systemically mediated anti-inflammatory activities.
Intravenously administered MSCs also significantly improved
LV function in mice with ischemic cardiomyopathy, changes
also accompanied by MSC-induced immunomodulatory effects. Our results therefore raise the possibility that intravenously administered MSCs may be a practical and effective
therapeutic strategy for the treatment of patients with large
infarcts and patients with ischemic cardiomyopathy. The validity of these concepts will have to await the results of appropriate clinical trials.
Sources of Funding
This study was supported by Medstar, Washington Hospital Center,
Medstar Health Research Institute, and CardioCell, LLC (San
Diego, CA).
Disclosures
S.E. Epstein is a consultant to and holds equity interest in CardioCell.
S. Sikora, A. Kharazi, and G. Vertelov are employees of CardioCell,
LLC.
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Intravenously Delivered Mesenchymal Stem Cells: Systemic Anti-Inflammatory Effects
Improve Left Ventricular Dysfunction in Acute Myocardial Infarction and Ischemic
Cardiomyopathy
Dror Luger, Michael J. Lipinski, Peter C. Westman, David K. Glover, Julien Dimastromatteo,
Juan C. Frias, M. Teresa Albelda, Sergey Sikora, Alex Kharazi, Grigory Vertelov, Ron
Waksman and Stephen E. Epstein
Circ Res. 2017;120:1598-1613; originally published online February 23, 2017;
doi: 10.1161/CIRCRESAHA.117.310599
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2017 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
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Supplement Material
Material and Methods
Acute myocardial infarction model:
CD1 male mice (8-10 weeks of age) were subjected to operative intervention to
create acute MI. Mice were anesthetized with sodium pentobarbital 70 mh/kg
administered by intaperitoneal injection and hair was removed from the left axilla. After
anesthesia, mice were intubated and ventilated with room air using positive pressure
respirator model 845 (Minivent, Harvard apparatus). Left thoracotomy was performed at
the fourth inter-costal space to expose the heart. Self-retaining microretractors were
used to separate the 3rd and 4th ribs enough to adequately expose the heart, while
leaving the ribs intact. After opening the pericardium, the left anterior descending
coronary artery (LAD) will be temporarily ligated with a 7-0 silk suture near its origin
between the pulmonary outflow tract and the edge of the left atrium. For the ischemia
reperfusion (IR) model, a 2-mm section of PE-10 tubing will be placed over the LAD and
the 7-0 silk suture will be tied into a slipknot on top of the tubing to occlude the LAD for
45 min (inducing ischemia). Ligation will be deemed successful when the anterior wall of
the left ventricle turns pale. The chest wall was approximated and covered with a piece
of moistened gauze to prevent desiccation during ischemia. Reperfusion was achieved
by removal of the PE-10 tubing. This allowed release of the occlusion and reperfusion of
the formerly ischemic myocardium. Cessation of positive pressure ventilation enabled
spontaneous inspiratory effort to fully inflate the lungs immediately prior to closing the
chest wall. The thoracotomy site was closed in layers using 5-0 prolene sutures for
closing the rib cage and then the skin. Animals were kept on a heating pad until they
recover after with they were housed individually in cages.
Human Mesenchymal Stem Cells
Primary frozen bone marrow derived human MSCs were received from
Stemedica (San Diego, Ca.) and cultured as described below. After expansion, cells
were aliquoted and frozen for future study. Culture conditions included: media
(DMEM/F12-GlutaMax (Invitrogen), HEPES Buffer 1X (Invitrogen), non-essential amino
acids 1X (Invitrogen), sodium pyruvate 1X (Invitrogen), fetal bovine serum 10% (Gibco),
antibiotic-antimycotic 1X (Gibco), 2 mercaptho-ethanol 1ul/ml (Sigma), fibroblast growth
factor (bFGF) 20 ng/ml (Shenandoah Biotechnology). Cells were cultured in 15 cm
dishes (Falcon). Cell confluence was achieved at 8-12 days, then cells were passed to
new dishes at 1X106 cells/dish. Cells from passage 3-6 were used for experiments.
Unless otherwise specified, 2 x106 MSCs in 0.2 ml were injected in each mouse into the
lateral tail vein 24 hours following AMI. In vitro experiments were performed utilizing
these cells incubated with murine serum and the degree of complement-mediated cell
lysis was assessed by determining cell viability.
Radiolabeling of MSCs
Molar excess of oxyquinoline (oxine) was incubated with indium-111 HCl
(PerkinElmer) for 30 minutes at room temperature in HEPES Buffer with 10% dimethyl
sulfoxide, enabling formation of indium-111 oxine. Indium-111 has a half-life of 2.8 days
with photon energies of 171 and 245 keV. MSCs were resuspended with indium-111
oxine solution and allowed to incubate at room temperature for one hour. Cells were
washed three times with normal saline after which percent cellular viability and MSC
counting was then determined by counting cells following Trypan Blue staining. Tail vein
injection of 1.0 x 106 radiolabeled MSCs was then performed 24 hours following AMI
(n=10) and in control mice without MI (n=10). The μCi of indium-111 per injected dose
was determined by measuring the radioactivity of the syringe before and following
injection into the mouse using a Radioisotope Dose Calibrator (Searle Radiographics).
Ex Vivo Nuclear Imaging of MSCs
Approximately 20 hours following intravenous injection of radiolabeled MSCs, the
recipient mice were euthanized by intraperitoneal injection of sodium pentobarbital
overdose, the vasculature was perfused with PBS, and the organs were harvested.
Short-axis cross-sections of the hearts were exposed to a high sensitivity, medium
resolution phosphor imaging screen (PerkinElmer). After 20 hours, the phosphor
imaging screen was scanned using a PerkinElmer Cyclone Plus Phosphor Imaging
System. Signal intensity analysis for each heart was performed in a blinded fashion
using Sante DICOM software and was adjusted for injected radioactive dose per animal
and background using the formula: (Mean Signal Intensity of heart - Background Signal
Intensity) / Injected Radioactive Dose in μCi). Signal intesity of the LAD region was
compared with the posterior wall to assess difference in signal intensity within the infarct
region. Comparison of MSC content based on indium-111 was then determined for
different organs using a Minaxi 5500 Gamma Counter using an indium-111 window
setting (160 to 485 keV). Gamma counting for each sample occurred over a five minute
period to generate an average number of counts per minute (CPM) and the adjusted
gamma counts for each organ was determined by the formula: ((CPM of tissue Background CPM) / Weight of the tissue in grams / Injected radioactive dose in μCi).
Ex-Vivo fluorescent Imaging
MSCs were fluorescently labeled by in vitro incubation for 30 minutes with Qdot
705 (Invitrogen) and injected as above. To identify tissue engrafted MSCs, mice were
sacrificed and organs were harvested; fluorescent optical imaging was performed with
an in-vivo imaging system (IVIS) 100 (Perkin Elmer, USA) at the relevant channel.
Echocardiograms
Echocardiograms were performed with a 30 mHz ultrasound probe (VEVO 1100
Imaging system, VisualSonics Inc) under light anesthesia with sodium pentobarbital (3040 mg/kg intraperitoneal injection). Short axis 2-dimensional echocardiogram images
were acquired at the midpapillary level of the LV and stored as digital loops. Anterior wall
thickness at end-systole (AWTs), LV end-systolic diameter, posterior wall thickness at
end-systole (PWTs), anterior wall thickness at end-diastole (AWTd), LV end-diastolic
diameter, and posterior wall thickness at end-diastole (PWTd) were calculated by
manual tracings of the endocardial border. These values were then utilized to calculate
LV ejection fraction (LVEF), end-systolic volume (LVESV), and end-diastolic volume
(LVEDV). Echocardiography was obtained at baseline prior to AMI surgery then at day 3,
7 and 20 post MI.
Preparation of single cell suspensions for flow cytometry
Single cell suspensions for flow cytometry and FACS were prepared in FACS
buffer. Mice were perfused with PBS through the LV to clear the blood system. Spleens
were mashed with the plunger of a 3ml syringe through a 70µm cell strainer (BD Falcon).
Heart tissue had been cut into 1mm pieces that were digested for 30 minutes with
collagenase II and DNAse. Single cell suspension was collected by mashing the
digested tissue through a 70µm cell strainer. Blood was obtained by cardiac puncture.
Blood samples were lysed twice in ACK buffer for leukocytes isolation. Cells were
recovered in complete medium, filtered through 40µm cell strainers, counted and
maintained on ice for further analysis.
Flow Cytometry
Cells surface Fc receptors were blocked by incubation with anti-Fc (CD32/CD16,
clone 93) (BioLegend, CA) for 15min. Cells were stained with the relevant antibodies in
FACS buffer for 30min at 4°C, washed and stained with 7-AAD to enable exclusion of
dead cells. Anti-mouse antibodies used for FACS staining (BioLegend, CA) were: CD19,
B220, CD5, CD3, CD4, CD8, CD11b, Ly6C, Ly6G, NKp46, DX5, NKG2D, CCR2,
CX3CR. Flow cytometry reading was done on FACS Calipur (BD CA.). Flow cytometry
analysis was performed with FlowJo (Ashland, OR).
TTC staining
Following the final echocardiogram, mice were administered a lethal dose of
sodium pentobarbital. Whole blood was harvested from mice at the time of sacrifice by
right ventricular puncture and the vasculature was perfused by LV puncture using PBS.
The heart was quickly excised, short-axis or axial cross-sections of the heart were cut to
1.0 mm thickness (Zivic Instruments, Pittsburgh, PA), and incubated at 37°C in 1% TTC
for 10 minutes. Heart sections were then fixed in 2% paraformaldehide overnight. Each
stained cardiac section was photographed and infarcted area was analyzed using Image
J software (NIH, Bethesda, MD). The areas were defined as follows: the infarct area
consists of the TTC-negative staining region (white/tan), and the non-ischemic region
consists of the TTC positively staining regions (red). Myocardial infarct size was
calculated as a percentage of the total left ventricle area from the slices distal to the
ligature.
In-vivo NK cells depletion
For in-vivo NK cells depletion the antibody anti-NK1.1 clone was injected
intravenously (100mg/ mouse) 24h prior to MI surgery. The experiment included a
control group (MI, n=12) and NK depletion group (MI + anti NK1.1, n=12).
Echocardiograms were measured at 7 and 21 days post MI. At 21 days post MI hearts
were analyzed by TTC staining for infarct size and the ratio of spleen immune cells was
analyzed by FACS. A number of mice were dedicated to validate in-vivo NK cells
depletion on day 7 post injection. NK depletion was evaluated by FACS analysis on
splenocytes.
Statistical Analysis
Continuous variables are presented as mean ± standard deviation unless
otherwise specified. Comparison of variables between the two groups was performed
using unpaired student T-test or Mann-Whitney U or Wilcoxon Rank-Sum Test in the
case of non-parametric data with non-normal distribution. Aspin-Welch test was utilized
in the case of unequal variance of the means. Correlation analysis was performed with
linear regression to provide a goodness of fit line and Pearson correlation coefficient.
Statistical analysis was performed with Number Crunching System 2007 software
(Kaysville, UT) and Graphpad Prism 6.0 software (San Diego, CA). A test was
considered statistically significant with p-value less than 0.05.
Online Discussion
Xenograft Model
When designing the current investigation, we made two important decisions
relating to the model. First, we decided to use human MSCs. The reason being that
mouse MSCs have many very different characteristics from human-derived MSCs1. We
were therefore concerned that the results we would have obtained with mouse MSCs
might not accurately reflect effects of human MSCs in human trials. This decision to use
human MSCs posed the dilemma of whether we had to use immunocompromised mice
to avoid the immune responses inherent in xenograft models. Use of
immunocompromised mice, however, would seriously limit the relevance of the results of
our studies, based as they are on the hypotheses that 1) the progressive LV dysfunction
observed in our mouse models of AMI and of ischemic cardiomyopathy was caused, at
least in part, by excessive immune/inflammatory activity, and 2) one of the important
mechanisms leading to possible beneficial effects of MSCs would derive from their
immunomodulatory effects.
However, use of a xenograft model—at least when MSCs are being use across
species--may in fact be possible. Jiang and colleagues conducted an extensive
literature search2 and identified 13 studies in which human MSCs were injected into
immunocompetent mice or rats. Each of these studies demonstrated by the time of study
termination (5 days to 6 weeks), depending on endpoints evaluated, either that MSCs
continued to be viable, or that the cells had contributed to positive outcomes. None of
these studies demonstrated evidence of rejection.
These results were interpreted as being consistent with the concept that MSCs
are immune privileged. However, a more nuanced perspective of the immune
“privileged” status MSCs has more recently evolved1. In their review, these authors
pointed out that administration of allogenic MSCs, or MSCs administered cross-species
(human MSC administered to mice), do not persist indefinitely; they postulated it is likely
that an active immunological process is responsible for this limited persistence.
Despite this lack of persistence, indicating that MSCs are not in reality immune
privileged, Ankrum and colleagues discussed the relative immune evasive capacity of
MSCs1. They presented the results of a study in which the relative persistence of allofibroblasts vs. allo-MSCs was compared. The study showed that fibroblasts died by day
10 and mMSCs by day 20—in other words, although the MSCs were not immune
privileged, they had some protection against immune rejection (the authors refer to this
as immune evasive) resulting in longer persistence than the non-MSCs. This concept
was also documented by using murine MSCs transfected with the gene expressing
erythropoietin as a reporter for persistent MSC functionality3. MSCs were injected
subcutaneously in either major histocompatibility complex (MHC)-mismatched allogeneic
or matched syngeneic mice. Although expression of erythropoietin lasted for a longer
time in the syngeneic mice, the mismatched MSCs still produced erythropoietin for over
30 days
These results suggest that although MSCs are not immune privileged and
eventually are eliminated from mismatched hosts through immune responses, they still
could exert therapeutic actions if the relevant activities of the MSCs need only a limited
time to produce their effects. Such a mechanism has been referred to as a “hit-andvanish” or “hit and run” mechanism1,4. Therefore, the question in either xenograft
models of disease or in the setting of administering allogenic MSCs to patients with
cardiac (or other) diseases becomes not one of “do MSCs have a total immune
privileged status” but, given their immune evasive capacity, “do they persist long enough
to exert a therapeutic effect”. Our data indicate that human MSCs persist for at least 20
days when administered IV into mice, and that this duration of persistence is sufficient to
lead to marked improvement in LV function.
Online Figure Legends
Online Figure I: Schematic for the experimental protocol
Acute MI Timeline.
Thirty-two male CD1 mice were randomized based on body weight and underwent
baseline echocardiograms (Blue arrow). Thereafter, mice underwent acute myocardial
infarction (AMI) surgery with 45 minutes of ischemia followed by reperfusion. At 24h
following MI treatment group were IV injected with MSCs (2x106/mouse) (green arrow)
and a control group were IV injected with the same volume of PBS (red arrow). Follow
up echocardiography were done on days 3, 7 and 20. At 21 days hearts were harvested
and analyzed for infarct size. Spleen cells were analyzed for the ratio of the different
immune cells. Echocardiograph measurements were further analyzed.
Ischemic Cardiomyopathy Timeline.
Thirty-two male CD1 mice underwent acute myocardial infarction (AMI) surgery with 45
minutes of ischemia followed by reperfusion. After 4 weeks the mice underwent baseline
echocardiography (blue arrows) with representative M-mode echocardiograms seen at
the top and bottom left. The top left echocardiogram demonstrates how measurements
were obtained for anterior and posterior wall thickness in systole (AWTs and PWTs),
anterior and posterior wall thickness in diastole (AWTd and PWTd), left ventricular endsystolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDD). The
representative baseline M-mode echocardiograms demonstrate evidence of prior MI with
reduced AWTs, increased LVESD and LVEDD, and decreased left ventricular ejection
fraction (LVEF). Mice were then randomized based on LVEF into two equal groups and
received either intravenous tail vein injection of 2.0 x 106 MSCs grown under hypoxic
conditions (green arrow) or PBS control (red arrow). The mice then underwent repeat
echocardiography at weeks 1 and 3 (blue arrows). The mice then underwent repeat
intravenous injection of MSCs (green arrow) or PBS control (red arrow). The mice then
underwent repeat echocardiography at weeks 4 and 6 (blue arrows), after which the
animals were euthanized and their organs were collected for analysis. Paired
representative M-mode echocardiograms for a mouse in the MSC treatment group (top
right) demonstrated improved systolic wall thickening with reduced LVESD and
increased LVEF while there was no improvement in the representative M-mode
echocardiogram at week 6 for the PBS control mouse.
Online Figure II: In vivo bio-distribution of murine MSCs in AMI
Radiolabeled murine MSCs grown under chronic hypoxia (5% O2) were injected
intravenously in mice 24h post-MI or in mice that underwent sham surgery,
(n=10/group). MSC content in different organs was measured 24h post-injection using a
gamma well counter. Heart and spleen MSC engraftment was calculated per gram tissue
(top panel). In the bottom panel, we provide comparison of trafficking of human and
murine MSCs to the heart and spleen 24h post injection using the gamma well counter in
mice that underwent AMI surgery 24 hours prior to injection.
Online Figure III: Intravenously-administered human MSCs improve cardiac
function in murine acute MI and ischemic cardiomyopathy models.
MSCs were administered 24h post-MI and their therapeutic effect was evaluated.
Echocardiography was performed at baseline prior to MI and then again at days 3, 7,
and 20 post-MI. Comparison is provided for anterior wall thickness in diastole (A),
posterior wall thickness in diastole (B), anterior wall thickness in systole (C), and
posterior wall thickness in systole (D) for the MSC treatment vs. control groups (n=16
per group). In the ischemic cardiomyopathy model (4 weeks post-MI), mice received
either intravenous MSC treatment or saline at baseline and week 3 (n=16 per group).
Serial echocardiography was performed at baseline, week 1, week 3, week 4, and week
6. Comparison is provided for anterior wall thickness in diastole (E), posterior wall
thickness in diastole (F), anterior wall thickness in systole (G), and posterior wall
thickness in systole (H) for the MSC treatment vs. control groups in the ischemic
cardiomyopathy model. *p<0.05, **p<0.01 in comparison to baseline.
Online Figure IV: Correlation of T lymphocyte populations with LV dimensions and
function
Flow cytometry analysis determined the T lymphocyte population (CD3+) and
subpopulations (CD4+ or CD8+), which were correlated with echocardiographic
parameters. Correlation matrices are presented plotting the splenic CD3+ cells as a
percentage of the total live splenocytes (A, B, and C), the splenic CD4+ cells as a
percentage of the total live splenocytes (D, E, and F), and the splenic CD8+ cells as a
percentage of the total live splenocytes (G, H, and I) on the X-axis. The Y-axis plots
LVEDV at 6 weeks (A, D, G), LVESV at 6 weeks (B, E, H), and LVEF at 6 weeks (C, F,
I). Data from the MSC treatment group (green circles) and from the PBS control group
(red squares) is plotted for each animal. Correlation analysis with best fit lines are
presented for each group with corresponding R2 value and p value. Among MSC
treatment mice, there was a correlation between higher T lymphocyte populations with
improved LV volumes and LV function (with the exception of G) while there was no
correlation among mice in the PBS control group.
References
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Ankrum JA, Ong JF, Karp JM. Mesenchymal stem cells: immune evasive, not
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