Supplemental data
Table 1.
1
Figure 1. Schematic representation of a stented vascular wall with the Genous stent capturing
circulating EPC.
Figure 2. Schematic extracorporeal shunt connection to arterial (red stopcock) and venous
(blue) catheter sheath introducer in femoral artery. Yellow squares indicate presence of 4
stents (2 BMS and 2 Genous stents) in the shunt tubing.
2
Figure 3. For the cell capture assay, a custom-made setup was created using sylgard 184
silicone tubes (with an inner diameter 3,14 mm, an a thickness of 0,5 mm). BM and Genous
stents were deployed to obtain a stent-tube ratio of 1,1 to 1,0. The labeled monocyte and
CD34+ cell suspensions were injected in the stented tube, and incubated under slow rotation
for 2 hours at a rate of 0,3 RPM at 37°C/5% CO2.
3
Supplemental material and methods
Scanning Electron Microscopy (human AV shunt)
The study stents were fixed in situ with 4% formalin/PBS, retrieved from the tubing, and
bisected longitudinally to expose the lumen surface. The stents were further processed for
scanning electron microscopy (SEM) and were rinsed in sodium phosphate buffer (0.1mmol/L, pH 7.2) and post-fixed in 1% osmium tetroxide for 30 minutes, followed by
dehydration in a graded series of ethanols. After critical point drying, the samples were
mounted and sputter-coated with gold. The specimens were visualized using a Hitachi Model
3600N scanning electron microscope and low power photographs of 10x were taken of the
lumen surface to estimate the degree of EC coverage of the implant. Regions of interest were
photographed at incremental magnifications of 200x to 600x.
mRNA processing and qPCR analysis
The captured cells were harvested from the stents by directly retrieving the RNA using a lysis
buffer provided by a commercial RNAeasy isolation kit, followed by cleanup using RNA
isolation columns (Qiagen, The Netherlands) according to the manufacture’s protocol as
conducted previously (1-3). Briefly; 20 ng carrier RNA was added to the cell lysate. 1 volume
of 70% ethanol was added to the homogenized lysate and the sample was mixed and isolated
by a commercial RNAeasy column. RNA was reverse transcribed using iScript reagents
according to the manufacturer’s protocol (Bio-Rad, The Netherlands). Gene expression was
quantified using qPCR by the SensiMix™ SYBR & Fluorescein Kit (Bioline, The
Netherlands), followed by signal detection on a MyiQ Single-Color Real-Time PCR
Detection System (BioRad, the Netherlands). Negative were included, whereas GAPDH and
beta actin served as housekeeping genes. Primers for the analysis were designed using the
4
sequences available on GenBank (http://www.ncbi.nlm.nih.gov/) and Primer3 software to
generate up to 3 primer pairs for each target sequence (Supplemental data table 1). All
primers were tested and the optimal primer pair was ultimately used for sample analysis.
Acute thrombogenicity
The thrombogenicity of Genous and BM stents was assessed using the ex vivo AV shunt
baboon model of arterial thrombogenicity(4), in which accumulation of Indium-labeled
platelets is measured in the stents implanted in a chronic arteriovenous femoral shunt in nonanticoagulated baboons. The baboons were not treated with anti-platelet drugs, but were
monitored daily for general health and CBC counts. All the animals included in our
experiments had normal clotting times as monitored weekly by aPT and PT.
After rinsing in sterile saline, the test stents were expanded in sterile tubing to 3.2 mm
diameter utilizing an inflation device and the sterile tubing was connected to the arteriovenous
loop and placed over a gamma camera. Platelet accumulation was measured at five minute
intervals by imaging
111
Indium labeled platelets with a gamma camera, for up to 2 hours
under continuous flow. The stents were subsequently visualized using scanning electron
microscopy, after radiation had dissipated(4).
In vitro assessment of human CD34+ cell capture and confocal microscopy
Human adult monocytes (advanced Biotechnologies, USA) were cultured in RPMI1640
medium (Gibco, USA) supplemented with 20% FCS at 37°C/5% CO2. Human peripheral
blood derived CD34+ cells (Allcells, USA) were expanded in expansion medium composed
of Stemline II Basal Media (Sigma, USA), supplemented with 2% G-CSF (Granulocyte CSF,
Sigma, USA), and 1% Stemspan Cytokine Cocktail (Stemcell Technologies, USA). Human
monocytes and CD34+ cells were labeled prior to the cell capture experiment, using a
5
fluorescent labeling kit according to the manufacturer’s instructions (PKH26 and PKH2 for
monocytes and CD34+ cells respectively, Sigma, USA). For the cell capture assay, a custommade setup was created using sylgard 184 silicone tubes (inner diameter 3,14 mm, 0,5 mm
thickness). BM and Genous stents were deployed to obtain a stent-tube ratio of 1,1 to 1,0 (see
supplemental figure 3). Labeled monocyte and CD34+ cell suspensions were injected in the
stented tube, and was incubated under slow rotation for 2 hours at a rate of 0,3 RPM at
37°C/5% CO2. After the rotation cycle, stents were fixed in 10% formalin for 10 minutes,
washed in PBS, cut longitudinally and imaged under confocal microscope (Zeiss, USA), using
490 nm/504 nm and 551 nm/567 nm excitation and emission for green fluorescently labeled
CD34+ cells and red fluorescently labeled monocytes respectively. Quantification of captured
cells was performed by counting individual cells on the struts, normalized to stent length.
Ex vivo human AV shunt
The study stents were introduced and deployed into a sterilized medical grade Silastic™
tubing (inner diameter 3,14 mm, 0,5 mm thickness, Dow Corning, USA,). BM and Genous
stents were deployed to obtain a stent-tube ratio of 1,1 to 1,0 and the shunt was connected
between arterial and venous sheaths that were placed in patients for vascular access to form an
AV shunt. The relative position of the BMS and the Genous stent in the AV shunt was
alternated to eliminate position bias. The patients were anti-coagulated with sodium heparin to
achieve an activated clotting time (ACT) greater than 300 seconds during their PCI procedure.
Additionally, the blood flow in the AV shunt was assessed continuously throughout the
experiments with a coronary flow wire to monitor for changes in blood flow indicative of
thrombotic occlusion in order to reduce the risk thrombo-emboli formation during the
procedure. The flow was maintained at 47.6 ± 14.6 cc/ml comparative to coronary flow.
6
Discussion
KDR is an EPC and EC marker, but is also a vital receptor for EC proliferation, implying that
the captured progenitor cells were also proliferative. Re-endothelialization efficiency of the
stent at a later time point was further studied in a rabbit model for arterial balloon injury and
vascular repair. Ultra-structural analysis demonstrated a modest increase in strut coverage by
spindle shaped (endothelial) cells at the proximal and distal ends of the Genous versus BM
stent, at 7 days after implantation. qPCR analysis subsequently validated the presence of EC
on the stent surface by mature endothelial (progenitor) cell markers including CD34, CD31,
Tie2, and P-selectin, as all these markers were upregulated in the EPC capture stent versus the
BMS group.
To further substantiate these findings, platelet aggregation was assessed in a baboon AV shunt
model. Accelerated re-endothelialization by CD34+ progenitor cells prevents platelet
adhesion in AV shunts in primates, potentially by competing with the adhesion of bloodderived platelets and inflammatory cells. Similarly, the adherent progenitor cell population
could contribute to stent surface protection against platelet aggregation(5, 6), rendering the
overall effect of the Genous stent to be vascular-protective. Additional in vitro experiments
using labeled human blood derived monocytes and CD34+ cells, clearly showed specific
capture of CD34+ cells on the Genous stent strut surface, whereas monocyte adhesion was
similar to that of BMS after a short cell-seeding procedure of 2 hours. This in vitro data would
suggest that the involved protective mechanism is not physical competition in cell adhesion,
but rather a potential paracrine effect of captured EPCs that benefits the prevention of platelet
aggregation.
To assess whether the stent coverage provides protection against inflammation in the injured
vessel segment, expression levels of CD16 was quantified. CD16 is expressed on neutrophils,
monocytes, macrophages and natural killer cells. When interpreting the data it has to be taken
7
into account that leukocyte adhesion during the human immune response is not a static
process. It includes cell activation, rolling, and adhesion, followed by either firm adhesion or
diapedesis over the basal membrane, or detachment from the vascular surface after which the
leukocyte re-enters the blood flow(7). Prolonged retention times on an activated vascular area
indicate an inflammatory response. CD16 levels correlated with the exposure of stents to
blood flow, showing a decrease in CD16 signal over time.
The efficacy of EPC capture was first assessed by conventional scanning electron microscope
(SEM) analysis, which revealed the presence of blood-derived cells with a rounded or flat
polygonal morphology indicative of EPC, and platelet aggregation on the stent struts. Semiquantitative analysis of the Genous stent revealed increase in cellular density (platelets were
excluded) on the stent surface as compared to BMS. To elucidate the identity of the captured
cells, surrogate biomarkers for EPC, EC, and immunocompetent cells and thrombogenicity
were assessed by qPCR analysis. Endothelial cell surface markers KDR, E-selectin, and
PLVAP mRNA were expressed in the attached cells in the Genous samples, as compared to
the BMS samples. However, only low expression levels of the CD34 marker were observed in
the lysates obtained from both stents. Limited CD34 expression in the EPC capture stent
sample could be due to active downregulation of CD34 in response to shear stress after cell
immobilization on the struts and exposure to blood flow leading into EC differentiation(8). It
is known that upregulation of EC markers and adaptation of the cultured EC morphology into
a more in vivo-like phenotype could be induced within minutes after exposure to mechanical
flow(9). In addition, loss of CD34 cell surface expression on circulating EPC is associated
with commitment to the endothelial linage. Furthermore, de Boer et al. has demonstrated that
in vivo platelet activation could rapidly induce differentiation of CD34+ cells into more
maturated KDR+ ECs(10). Therefore, the low level of CD34 expression on the captured cells
could be explained by rapid differentiation of the progenitor cells towards a mature EC type.
8
In line with this hypothesis, expression of more mature EC markers, including E-selectin were
indeed upregulated in the Genous stent samples as compared to BMS. For an extended
discussion of the qPCR data, see supplemented data.
9
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