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ONLINE APPENDIX
ONLINE SUPPLEMENTAL METHODS
MICE
Eight to ten weeks old C57BL/6J (stock number: 000664) male mice were
purchased from Jackson Laboratory. All experiments conform to protocols approved by
the Institutional Animal Care and Use Committee at Northwestern University (Chicago,
IL) and Temple University (Philadelphia, PA).
MOUSE BONE MARROW CELL ISOLATION AND EPC CULTURE
Bone marrow-EPC isolation, ex vivo expansion and culture of EPCs were
performed as previously described (1). In brief, bone marrow mononuclear cells were
isolated from mice by density-gradient centrifugation with Histopaque-1083 (Sigma) and
macrophage-depleted by allowing attachment to uncoated plate for 1 h. The unattached
cells were removed and plated on culture dishes coated with 5µg/ml human fibronectin
(Sigma) and cultured in phenol red–free endothelial cell basal medium-2 (EBM-2,Lonza)
supplemented with 5% FBS and EGM-2 MV SingleQuots (Lonza) containing VEGF,
fibroblast growth factor-2, epidermal growth factor, insulin-like growth factor-1, ascorbic
acid, and antibiotics. Cells were cultured at 37°C with 5% CO 2 in a humidified
atmosphere. After 4 days in culture, non-adherent cells were removed by washing with
PBS, followed by addition of fresh media and the culture was maintained through day 7.
EPCs, recognized as attaching spindle-shaped cells were used for further analysis and
treatment.
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ISOLATION OF ADULT MOUSE CARDIOMYOCYTES
The protocol for mouse adult cardiomyocyte isolation has been modified and is
previously described and has been modified from (2,3). Briefly, 10- to 12-week-old
(C57BL/6J) mice were heparinized (50 USP units) and anesthetized. The heart was
quickly removed from the mice, cannulated through the ascending aorta, and mounted
on a Langendorff perfusion apparatus. The heart was then retrogradely perfused (3
mL/min) for 5 min at 37ᵒC and constant pressure with pre-filtered Ca2+-free bicarbonatebased buffer containing 120 mM NaCl, 5.4 mM KCl, 1.2 mM MgSO4, 1.2 mM NaH2PO4,
5.6 mM glucose, 20 mM NaHCO3, 10 mM 2,3-butanedione monoxime (BDM; Sigma),
and 5 mM taurine (Sigma), gassed with 95% O2–5% CO2. The enzymatic tissue
digestion was initiated by adding collagenase type B (0.5 mg/ml; Boehringer Manheim),
collagenase type D (0.5 mg/ml; Boehringer Manheim), and protease type XIV (0.02
mg/ml; Sigma) to the perfusion solution for 3-5min, after which 50 μM Ca2+ was added
to the enzyme solution. Once the heart becomes swollen and yellowish in color it was
quickly removed from the apparatus and manually separated into small pieces for
further digestion (in a shaking water bath for 10 min at 37°C) in the same enzyme
solution. The suspension containing the dispersed myocytes was filtered through 100
µm filter and transferred to stopping buffer (perfusion buffer with the addition of 10%
FBS) and gently centrifuged at 500 rpm for 30 sec. Cardiomyocyte Ca2+ reintroduction
and cell purification was performed by adding the cells to progressively increased CaCl 2
buffer concentrations (125 µM, 250 µM, 500 µM) and allowed to gravity sediment in 10
ml for 10 min each.
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HUMAN CD34+ CELLS CULTURING AND TRANSIENT TRANSFECTION WITH
MiRNA MIMIC AND INHIBITOR
hCD34+ cells were cultured in XVIVO (Lonza) media supplemented with
StemSpan™ CD34+ Expansion Supplement (10X) and 0.5% human serum albumin as
described in our previous report (4). hsa-miR-377 mimic [mirVanaTMmiRNA mimic
Cat#:4464066, (Ambion, Life Technologies)] or hsa-miR-377 inhibitor (mirVanaTMmiRNA
inhibitor Cat#:4464084), and their respective nonspecific control (mirVanaTMmiRNA
mimic negative control, Cat#:4464058) or mirVanaTMmiRNA inhibitor negative control
(Cat#:4464066) were used for transfection. miRNA mimics are small, chemically
modified double-stranded RNA molecules designed to specifically bind to and mimic
endogenous miRNA molecules and enable miRNA functional analysis by up-regulation
of miRNA activity. miRNA inhibitors are small, chemically modified single-stranded RNA
molecules designed to specifically bind to and inhibit endogenous miRNA molecules
and enable miRNA functional analysis by down-regulation of miRNA activity. miRNA
mimic or miRNA inhibitor negative control are random sequence miRNA mimic or
miRNA inhibitor molecule that has been extensively tested in human cell lines and
tissues and validated to produce no identifiable effects on known miRNA function.
hCD34+ cells were seeded in 6-well plates 24 h prior to transfection. The miR377-mimic and inhibitor were added at the final concentration of 60 nM after mixing with
Lipofectamine transfection reagent (Invitrogen) according to the manufacturer’s
instructions. After 48 h, the cells from each group were harvested for PCR and Western
blot analysis.
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RNA ISOLATION AND QUANTITATIVE REVERSE TRANSCRIPTION PCR
Briefly, cells or left ventricular tissue was homogenized using lysis reagent and
the homogenized samples were incubated at room temperature for 5 min. RNA was
isolated using miRNeasy Mini Kit (Qiagen). First strand cDNA was synthesized using a
TaqMan MiRNA Reverse Transcription Kit by following the vendor’s instructions
(Applied Biosystems, Foster City, CA). qRT-PCR was performed using the Step One
Plus system (Life Technologies) following the manufacturer’s instructions. Following
cDNA synthesis, amplification was performed using Taqman 7300 (Applied Biosystems,
Foster City, CA). Relative miRNA or mRNA expression of target gene was normalized
to the U6 control or GAPDH gene, respectively (4).
PROTEIN ISOLATION AND WESTERN BLOT ANALYSIS
Protein isolation and Western blotting for cultured cells was performed as
previously described (4,5). Briefly, cells were homogenized in lysis buffer (Cell Signaling
Technology, MA, USA) containing 20 mmol/l Tris-HCl [pH 7.5], 150 mmol/l NaCl, 2.5
mmol/l sodium pyrophosphate, 1 mmol/l β-glycerophosphate, 1 mmol/l sodium
orthovanadate, 1 μg/ml leupeptin, 1 mmol/l ethylenediaminetetraacetic acid [EDTA], 1
mmol/l ethylene glycol tetraacetic acid [EGTA], 1% Triton X-100 and protease inhibitors.
Equal amounts of protein were separated by 10% SDS-PAGE and blotted onto
polyvinylidenedifluoride (PVDF) membranes (Bio-Rad, Hercules, CA). The blots were
incubated with antibodies against STK35 (Abcam, USA) and developed with an
enhanced chemiluminescence detection system (Amersham, Piscataway, NJ).
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HUVEC MIGRATION ASSAY
The migration of HUVEC transfected with miR-377 mimic or miR-377 inhibitor or
respective negative control toward stimulation (VEGF) was performed in a 24-well Trans
well Chamber as described earlier (5). Briefly, 20 ng/ml VEGF in 600 μl of chemotaxis
buffer (serum-free EBM-2, 0.1% BSA) was added to the lower compartment. HUVEC
(1×105 cells) in 100 μl of chemotaxis buffer were added to the upper compartment. After
incubation at 37°C for 18 h, the filters were removed and the cells that migrated through
the pores to the bottom chamber were stained with Hema-3® stain kit (Millipore,
Billerica, MA) and counted manually in random high-power fields (~200) in each well.
Data are expressed as number of cells that migrated through or invaded pores. All
groups were studied at least in triplicate.
VASCULAR TUBE FORMATION ASSAY
HUVECs were transfected with miR-377 mimic or miR-377 inhibitor or respective
negative control for 48 h. The media supernatant was collected and mixed in equal
quantities (1:1) and added to growth factor reduced EBM-2 media with 2% FBS (Lonza,
Basel, Switzerland) to 1.5 × 104 HUVECs plated on 40 µl Matrigel (BD Falcon) in a 48
well plate. After incubation (4-6 h) at 37°C in an atmosphere of 5% CO2 plates were
observed by using a phase contrast microscope. The cumulative tube length was
measured in each field. Results are represented as s.e.m for three independent
experiments.
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ECHOCARDIOGRAPHY
Transthoracic two-dimensional M-mode echocardiogram was obtained using
Vevo 770 (VisualSonics, Toronto, Canada) equipped with a 30 MHz transducer.
Echocardiographic studies were performed before (baseline) and at 28 days post-IR on
mice anesthetized with a mixture of 1.5% isoflurane and oxygen (1 l/min). M-mode
tracings were used to measure end-systolic diameter (LVESD) and end-diastolic
diameter (LVEDD) and percent ejection fraction (%EF) was calculated as described
previously (5).
CARDIAC FIBROSIS ASSESSMENT
Histopathology studies were performed as described previously (4,5). The hearts
were fixed with 10% buffered formalin and paraffin embedded. Cardiac fibrosis area
was quantified on Masson’s trichrome-stained sections by using NIH’s Image J software
to determine percent fibrosis.
MYOCARDIAL CAPILLARY DENSITY
Immunofluorescence staining for tissue sections were performed as described
previously (5). Tissue sections were permeabilized and stained with anti-human CD31
antibody (Cat#: ab28364, Abcam). Nuclei were counter-stained with 4', 6-diamidino-2phenylindole (DAPI, 1:5000, Sigma Aldrich, St Louis, MO), and sections were examined
with a fluorescent microscope (Nikon ECLIPSE TE200, Japan). The number of CD31+
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were assessed in 10 randomly selected high-power visual fields (HVF) and expressed
as number per HVF.
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References
1.
Yamaguchi J, Kusano KF, Masuo O et al. Stromal cell-derived factor-1 effects on
ex vivo expanded endothelial progenitor cell recruitment for ischemic
neovascularization. Circulation 2003;107:1322-8.
2.
Zhou YY, Wang SQ, Zhu WZ et al. Culture and adenoviral infection of adult
mouse cardiac myocytes: methods for cellular genetic physiology. American
journal of physiology Heart and circulatory physiology 2000;279:H429-36.
3.
Garcia-Prieto J, Garcia-Ruiz JM, Sanz-Rosa D et al. beta3 adrenergic receptor
selective stimulation during ischemia/reperfusion improves cardiac function in
translational models through inhibition of mPTP opening in cardiomyocytes.
Basic research in cardiology 2014;109:422.
4.
Thal MA, Krishnamurthy P, Mackie AR et al. Enhanced angiogenic and
cardiomyocyte differentiation capacity of epigenetically reprogrammed mouse
and human endothelial progenitor cells augments their efficacy for ischemic
myocardial repair. Circulation research 2012;111:180-90.
5.
Krishnamurthy P, Thal M, Verma S et al. Interleukin-10 deficiency impairs bone
marrow-derived endothelial progenitor cell survival and function in ischemic
myocardium. Circulation research 2011;109:1280-9.
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ONLINE FIGURES AND LEGENDS
Online Figure 1. MiR-377 Expression in LPS Treated Mouse EPC and HUVECs.
MiR-377 expression in the control and LPS treated (A) mouse EPC and (B) HUVEC
were validated by qRT-PCR (normalized to control U6, n=6, *P < 0.05).
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Online Figure 2. Heat Map Showing Differentially Expressed MiRNAs in LPS
Treated Mouse EPC.
EPCs were treated with LPS (25 ng/ml) for 12 hr and miRNA expression was analyzed
using PCR-based miRNA microarray platform covering a total of 352 mouse miRNAs.
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Online Figure 3. MiR-377 Expression in MiR-377 Mimic, Inhibitor or miR Mimic or
Inhibitor Negative Controls Treated hCD34+ Cells and HUVECs.
(A) hCD34+ cells and (B) HUVECs were transfected with miR-377 mimic, inhibitor or
miR mimic or inhibitor negative controls and were subject to qRT-PCR. Data was
normalized to internal U6 control and expressed as fold change vs respective negative
controls. n=6, (*P<0.05, miR-377 mimic vs miR mimic negative control; #P<0.05, miR377 inhibitor vs miR inhibitor negative control).
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Online Figure 4. Presence of Transplanted miR Mimic Negative Control and MiR377 Knockdown hCD34+ Cells Labelled with PKH26 in the Mice Myocardium at 3
Day After IR.
(A, B) PKH26+ labelled hCD34+ cells after transplantation into the mice myocardium
HCD34+ cells (PKH26 positive, red florescence) and DAPI (blue) for nuclear staining.
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Online Figure 5. Effect of VEGF on MiR-377 Expression.
(A) HUVECs were treated with VEGF and miR-377 expression was determined by qRTPCR (normalized to control U6, n=6). (B) HUVECs were transfected with miR-377 mimic
or Inhibitor or miR mimic or inhibitor negative controls and VEGF mRNA level was
determined by qRT-PCR. (n=6, ¶P<0.05, VFGF treated vs control untreated: *P<0.05,
miR-377 mimic vs miR mimic negative control; #P<0.05, miR-377 inhibitor vs miR
inhibitor negative control).