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Online Appendix for the following JACC article
TITLE: Intramyocardial Injection of Platelet Gel Promotes Endogenous Repair and
Augments Cardiac Function in Rats With Myocardial Infarction
AUTHORS: Ke Cheng, PHD, Konstantinos Malliaras, MD, Deliang Shen, MD, Eleni
Tseliou, MD, Vittoria Ionta, PHD, Jeremy Smith, MS, Giselle Galang, MS, Baiming Sun,
MD, Christiane Houde, MS, Eduardo Marbán, MD, PHD
APPENDIX
Supplementary Information
Expanded Methods
Derivation of platelet gel
Platelet gel was derived from venous blood of Wistar-Kyoto (WKY) or Sprague-Dawley
(SD) rats according to previously reported methods (1). Briefly, deep anesthesia was
introduced by inhalation of isoflurane. After that, the rat’s abdominal skin was dissected
and blood was drawn from the vena cava. The blood was immediately citrated with 10%
(v/v) 10 mM sodium citrate (Sigma-Aldrich, St Louise, MO). Whole blood samples were
then centrifuged at 1,000g for 10 min and the supernatant collected. The plateletcontaining plasma was then collected and mixed with pre-warmed DMEM at a ratio of
1:1 (v/v) for gel formation. To visualize the fibrous structure and presence of platelets,
the formed platelet gel was frozen, cryosectioned, and subjected to H&E staining. A
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stress-controlled shear rheometer (AR-2000, TA Instruments) with 20-mm parallel disc
geometry was used to evaluate the rheological properties of the gelation process.
To enable histological detection of injected platelet gel in vivo, we labeled the fibrin
components by incubation with Texas Red-X succimidyl ester (1 mg/ml; Invitrogen) for
30 min at 37°C immediately before gelation.
In vitro cytokine release
A 150 µl platelet gel was formed in each well of a 24-well plate and incubated with 1 ml
of FBS-free Iscove's Modified Dulbecco's Medium (IMDM; Invitrogen). To study
sustained release of cytokines and growth factors, the conditioned media was collected at
various time points (days 2, 5, 9, and 14), and fresh media was added back into the well
to be conditioned for the next time point. The concentrations of VEGF, IGF-1, and HGF
in the conditioned media were measured by rat-specific ELISA kits (R&D Systems,
Minneapolis, MN; B-Bridge International, Cupertino, CA), according to the
manufacturer's instructions.
NRCM culture
To test if the platelet gel is compatible with cardiomyocytes, we cultured neonatal rat
cardiomyocytes (NRCM) from SD rats in platelet gel derived from the same strain of
animals. NRCMs were isolated by routine methods with overnight trypsin digestion from
neonatal SD rats and plated confluently on fibronectin-coated multiwells in Medium 199,
10% for 48 h, then 2% FBS, 10 mmol/l HEPES, 0.1 mmol/l MEM nonessential amino
acids, 19.4 mmol/l glucose, 2 mmol/l L-glutamine, 0.8 µg/ml vitamin B12, and 2 unit/ml
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penicillin. To culture NRCM in the platelet gel, the NRCM-containing DMEM was
mixed with the platelet-containing plasma to form stable gels. The culture was incubated
at 37ºC and 5% CO2. The cells were stained with CM-DiI or Calcein-AM/EthD
(Invitrogen) for assessment of morphology and viability. Briefly, 200,000 NRCMs were
resuspended in 200 µl of platelet gel in one well of a 24-well plate. The control is the
same number of cells seeded into the well (without platelet gel). After 14 days in culture,
the specimens were examined for live and dead cells.
Animal model
Animal care was in accordance with Cedars-Sinai Medical Center Institutional Animal
Care and Use Committee (IACUC) guidelines. Platelet gel was derived from male WKY
rats and then intramyocardially injected into female WKY rats (n = 78 total) via a left
thoracotomy under general anesthesia, and myocardial infarction (MI) was produced by
permanent ligation of the left anterior descending (LAD) coronary artery. Briefly, a
sterile suture was used to permanently occlude the LAD approximately 1 mm distal to the
edge of the left atrium to create a medium-sized infarct typically involving ~30% of the
LV. Immediately after MI, the animals were subjected to intramyocardial injections with
a 29G needle at 4 points in the infarct zone, under 1 of 2 randomly assigned conditions:
1) control group: injection of 150 µl of vehicle (DMEM); and 2) gel group: injection of
150 µl of platelet gel. For platelet gel injection, 75 µl of host plasma were mixed with 75
µl of pre-warmed DMEM. Because gel formation happened quickly, the mixture was
immediately drawn into a syringe and injected into the myocardium.
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Morphometric analysis
For morphometric analysis, animals were euthanized at 3 weeks and the hearts were
harvested and frozen in OCT compound. Sections every 100 µm (10 µm thickness) were
prepared. Masson’s trichrome staining (6 sections per heart, collected at 400 μm
intervals) was performed as described (2). Images were acquired with a PathScan Enabler
IV slide scanner (Advanced Imaging Concepts, Princeton, NJ). From the Masson’s
trichrome-stained images, morphometric parameters including LV cavity area, infarct
wall thickness and infarct perimeter were measured in each section with NIH ImageJ
software. Six measurements were averaged for each heart.
Histology
Heart cryosections were fixed with 4% PFA, permeabilized/blocked with Protein Block
Solution (DAKO, Carpinteria, CA) contains 1% saponin (Sigma, St. Louis, MO), and
then stained with combinations of the following antibodies: mouse monoclonal antiCD68 [ED1] (Abcam, Cambridge, MA), rabbit polyclonal anti-von Willebrand factor
(Abcam), rabbit polyclonal anti-ckit (Abcam), mouse monoclonal anti-alpha sarcomeric
actin [EA-53] (Sigma), and rabbit polyclonal anti-laminin (Abcam) antibodies. FITC or
Texas Red secondary antibodies were used for detection. Capillaries were stained with
FITC-conjugated isolectin B4 (Vector Lab). Images were taken by a Leica TCS SP5 X
confocal microscopy system (3 sections per heart, collected at 800 μm intervals). In each
section, 3 randomly selected fields in the infarct area were selected for quantitation. The
counts were averaged for each heart. The degradation of platelet gel was quantified by
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measuring the myocardial area covered by Texas Red labeled gel, using NIH ImageJ
software.
Cardiac function assessment
To assess global cardiac function, echocardiography was performed with the Vevo 770
system (Visual Sonics, Toronto, Ontario, Canada) on day 0 (baseline; 4 hours post-MI),
and 3 and 6 weeks afterward. The LV size was measured from the parasternal long-axis
view. The LVEF was calculated with Visual Sonics V1.3.8 software from 2D long-axis
views taken through the infarcted area. Both absolute values and changes from baseline
(day 0 post-MI) are reported. Blinded reading of echos was conducted independently by
an experienced echocardiographer (D.S.).
Pathway-focused PCR array
Using the RT2 Profiler PCR Array System (SABiosciences Corporation, Frederick, MD),
we compared gene expression of angiogenesis in control- and platelet gel-treated hearts at
3 weeks. The array consists of more than 84 angiogenesis-related genes with gene
expression quantified using real-time PCR, according to the manufacturer's instructions.
Briefly, total RNA from myocardium samples was extracted using RNeasy Micro Kit
(Qiagen). The cDNA was prepared from the total RNA mixture of six independent
patients using the RT2 First Strand Kit (SABiosiences). The experimental cocktail was
prepared by adding cDNA to RT2 qPCR Master Mix (SABiosiences) within the 96-well
PCR array. Real-time PCR was performed in 7900HT Fast real-time PCR System. Data
were analyzed using the 7900HT Sequence Detection System Software v2.3 (Applied
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Biosystems) and PCR Array Data Analysis Software (SABiosiences). As a quality
control measure, threshold cycle <30 was used as the criteria for including a gene in the
data.
Assessment of microvascular perfusion with lectin angiography
To ensure that newly formed micro-vessels are functionally perfused, a subgroup of
animals underwent lectin microvascular angiography as described (3). Briefly, 500 µg/kg
of fluorescein-labeled Lycopersicon esculentum (tomato) lectin (Vector Laboratories)
was injected into the inferior vena cava. Five minutes after lectin perfusion, the animals
were sacrificed and the hearts were explanted. Image stacks were obtained with confocal
microscopy through 100-µm-thick heart sections of the infarct zone (3 sections per heart,
collected at 800-μm intervals). The green fluorescence was quantified with NIH ImageJ
and the values were normalized to those from noninfarcted normal hearts to generate the
relative blood flow data.
Tissue Western blot analysis and inflammatory cytokines
Myocardial samples from the peri-infarct area were collected 7 days post-MI. Tissue
samples were lysed in lysis buffer supplemented with proteinase inhibitor cocktail
(Roche, MN) and homogenized with a rotor-stator homogenizer (Eppendorf, Hauppauge,
NY). Homogenates were centrifuged at 10,000 rcf for 10 minutes on ice, supernatants
were collected, and protein content was quantified by Lowry assay (BioRad, Hercules,
CA). The equivalent of 15 µg of total protein per lane was loaded onto 12% Precise
Protein gels (Thermo Scientific, Rockford, IL), and then transferred to PVDF
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membranes. Membranes were blocked with 5% nonfat milk and incubated overnight with
primary antibodies against tumor necrosis factor-alpha (TNF-alpha) and GAPDH
(Abcam). Subsequently, the appropriate HRP-conjugated secondary antibodies were
used, and then the blots were visualized by using SuperSignal West Femto substrate
(Thermo Scientific) and exposed to Gel Doc XR System (Bio-Rad). Quantitative analysis
was performed by ImageJ software, and expressions were normalized to GAPDH.
Enzymatic isolation of cardiomyocytes for assessment of hypertrophy
Cardiomyocytes were isolated from WKY rats 3 weeks after control or platelet-gel
treatment by enzymatic dissociation of the whole heart on a Langendorff apparatus as
described (4). Heparinized animals were anesthetized by ketamine/xylazine (30 mg/kg
and 6 mg/kg, respectively). Hearts were rapidly excised and washed of blood in ice-cold
Tyrode's solution before being mounted to a Langendorff apparatus conjugated to a
pressure monitoring device, then perfused retrogradely with the following four
oxygenated solutions in sequential order: 1) modified Tyrode's solution containing 1.0
mM Ca2+ (2 min); 2) Ca2+-free Tyrode's solution (2–3 min); 3) Ca2+-free Tyrode's
solution containing 0.2 Wünsch unit/ml of collagenase made from Liberase Blendzyme 4
(Roche Molecular Biochemicals, Indianapolis, IN) for 10–15 min depending on digesting
conditions; and 4) washed in Kruftbrühe (KB) solution for 5 min. Digested atria and
ventricles were cut off and minced in KB solution, pipetted to dissociate the cells, then
filtered through a nylon mesh (200-µm pore size) to remove big pieces of undigested
tissue. Isolated cells were rinsed in KB solution and allowed to settle by gravity 3 times
to remove debris and noncardiomyocytes. Cells resuspended in KB solution were loaded
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above the top layer of a Percoll gradient formed by 20%, 40%, and 70% Percoll and
centrifuged at 100g for 20 min to further purify cardiomyocytes. After three washes in
KB solution, myocytes were resuspended in KB solution or in culture media for further
experiments. The sizes of myocytes were evaluated in cells cytospun onto 22-mm cover
glasses by fluorescent immunocytochemistry (anti–alpha-sarcomeric actin) in
combination with confocal microscopy.
Statistical analysis
Results are presented as mean ± SD unless specified otherwise. Statistical significance
between baseline and 3-week LVEFs was determined using 2-tailed paired Student’s t
test. All the other comparisons between any 2 groups were performed using 2-tailed
unpaired Student’s t test. Differences were considered statistically significant when p <
0.05.
References
1. Bryan N, Rhodes NP, Hunt JA. Derivation and performance of an entirely autologous
injectable hydrogel delivery system for cell-based therapies. Biomaterials
2009;30:180–8.
2. Cheng K, Li TS, Malliaras K. Magnetic targeting enhances engraftment and
functional benefit of iron-labeled cardiosphere-derived cells in myocardial infarction.
Circ Res 2010;106:1570–81.
3. Frederick JR, Fitzpatrick JR III, McCormick RC, et al. Stromal cell-derived factor1alpha activation of tissue-engineered endothelial progenitor cell matrix enhances
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ventricular function after myocardial infarction by inducing neovasculogenesis.
Circulation 2010;122:S107–17.
4. Zhang Y, Li T-S, Lee S-T, Wawrowsky KA, et al. Dedifferentiation and proliferation
of mammalian cardiomyocytes. PLoS ONE 2010;5:e12559.
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Supplementary Figures
Supplementary Figure 1. Viability of NRCM cultured in platelet gel. (A) Fluorescent
micrographs showing Calcein-AM (live)/EthD (dead) staining of NRCMs cultured in
platelet gel for 14 days. Most of the cells were viable. Only few cells were stained with
EthD (while arrows). (B) Quantification of viable NRCMs cultured in platelet gel and
conventional tissue culture surface. Experiments were run in triplicates. Bar = 50 µm.
NRCM = neonatal rat cardiomyocytes.
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Supplementary Figure 2. Platelet gel reduces apoptosis, recruits c-kit-positive stem
cells, but does not exacerbate inflammation in the infarct. (A, B) Apoptotic cells 7
days after control or platelet gel injection were identified by TUNEL staining. (C)
Quantitation of apoptotic cells in the control- and gel-treated hearts. (D, E)
Representative images showing c-kit-positive cells in the control- and gel-injected hearts
at Day 7 after injection. More c-kit cells were evident in the gel-treated hearts. Some cells
had penetrated into the gel (E, white arrows). (F) Quantitation of c-kit-positive cells in
the control- and gel-treated hearts. (G, H) Fluorescent microscopy images showing the
presence of CD68-positive macrophages in the infarct area at Day 7 after injection. No
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macrophage infiltration into the gel was found. (I) Quantitation of macrophages in the
infarct area from control- and gel-treated hearts. Bas = 100 um.* indicates p < 0.01 when
compared to Control.
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Supplementary Figure 3. Tissue Western blot analysis of tumor necrosis factoralpha (TNF-alpha) in gel- and control-treated hearts. (A) Representative Western blot
bands showing the expressions of TNF-alpha and house keeping gene GAPDH. (B)
Quantification of TNF-alpha expressions (normalized to GAPDH).
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Supplementary Figure 4. Platelet gel reduces myocyte hypertrophy and increases
capillary density. (A, B) Representative confocal microscopy images of cardiomyocytes
in the normal region stained with alpha-SA (cell body) and laminin (cell boundary). (C)
Quantitation of myocyte cross-section areas from the control- and gel-treated hearts. (D,
E) Representative confocal microscopy images of capillary structures stained with FITCconjugated isolectin B4. (F) Quantitation of capillary densities from the control- and geltreated hearts. Bars = 30 um. * indicates p < 0.001 when compared to Control.
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Supplementary Figure 5. Enzymatic isolation of cardiomyocytes for assessment of
hypertrophy. (A, B) Enzymatically isolated cardiomyocytes from control- or platelet
gel-treated hearts (3 weeks post-treatment) Red, alpha-sarcomeric actin. (C)
Quantification of myocyte area by Leica LAS AF software. *Indicates p < 0.05 when
compared to Control. Bar = 100 µm.
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Supplementary Figure 6. Lectin microvascular angiography. (A, B) Confocal images
showing lectin-labeled blood vessels in control- or gel-treated hearts (n=3 hearts per
group) at 3 weeks. (C) Lectin microvascular angiography in a normal heart (no MI). (D)
Quantification of lectin fluorescence (relative to that from the normal heart) in controland gel-treated animals. *Indicates p < 0.05. Bars = 200 µm.
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Supplementary Figure 7. Angiogenesis pathway-focused PCR analysis. The fold
change of gel-treated hearts relative to control-treated hearts was reported (n=2).
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