Exploring the use of the Suzuki coupling reaction in the synthesis of 4′-alkyl-2′-hydroxyacetophenones

Article abstract of DOI:10.1055/s-0033-1340312

A series of 4′-alkyl-2′-hydroxyacetophenones were prepared by Suzuki cross-coupling reactions of 4′-bromo-2′-hydroxyacetophenone. In these reactions, alkyl(trifluoro)borates were found to be better reactants than alkylboronic acids. 4′-Alkyl-2′-hydroxyacetophenones are key intermediates for the further synthesis of ?lipoflavonoids that are more readily incorporated into lipid bilayer membranes than flavonoids and should, therefore, have superior ?biological effects through increased bioavailability. Georg Thieme Verlag Stuttgart New York.

Full text of DOI:10.1055/s-0033-1340312

European Heart Journal Advance Access published March 25, 2013
BASIC SCIENCE PAPER
European Heart Journal
doi:10.1093/eurheartj/eht105
miRNA-21 is dysregulated in response to vein
grafting in multiple models and genetic ablation
in mice attenuates neointima formation
Robert A. McDonald1, Katie M. White1, Junxi Wu2, Brian C. Cooley3,
Keith E. Robertson1, Crawford A. Halliday1, John D. McClure1, Sheila Francis4,
Ruifaug Lu1, Simon Kennedy1, Sarah J. George5, Song Wan6, Eva van Rooij7,
and Andrew H. Baker1
*
1Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow G12 8TA, UK; 2Strathclyde Institute of Pharmacy and Biomedical Sciences, University of
Strathclyde, Glasgow G40NR, UK; 3Department of Orthopaedic Surgery, Medical College of Wisconsin, Milwaukee, WI, USA; 4Department of Cardiovascular Science, Medical
School, Sheffield S10 2RX, UK; 5Bristol Heart Institute, School of Clinical Sciences, Research Floor Level 7, Bristol Royal Infirmary, Bristol BS2 8HW, UK; 6Division of Cardiothoracic
Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China; and 7Hubrecht Institute, KNAW and University Medical Center, Utrecht,
the Netherlands
Received 25 January 2013; revised 5 March 2013; accepted 6 March 2013
Aims
The long-term failure of autologous saphenous vein bypass grafts due to neointimal thickening is a major clinical
burden. Identifying novel strategies to prevent neointimal thickening is important. Thus, this study aimed to identify
microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological
relevance following miRNA manipulation.
.....................................................................................................................................................................................
Methods
and results
We undertook a microarray approach to identify dysregulated miRNAs following engraftment in an interpositional
porcine graft model. These profiling experiments identified a number of miRNAs which were dysregulated following
engraftment. miR-21 levels were substantially elevated following engraftment and these results were confirmed by
quantitative real-time PCR in mouse, pig, and human models of vein graft neointimal formation. Genetic ablation
of miR-21 in mice or grafted veins dramatically reduced neointimal formation in a mouse model of vein grafting. Fur-
thermore, pharmacological knockdown of miR-21 in human veins resulted in target gene de-repression and a signifi-
cant reduction in neointimal formation.
.....................................................................................................................................................................................
Conclusion
This is the first report demonstrating that miR-21 plays a pathological role in vein graft failure. Furthermore, we also
provided evidence that knockdown of miR-21 has therapeutic potential for the prevention of pathological vein graft
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Keywords
Vein graft failure † MicroRNA † Neointimal formation † Vascular remodelling
A number of studies have demonstrated that patency rates of
saphenous vein (SV) grafts are lower than those of arterial conduits
such as the internal mammary artery,4,5 yet autologous veins
remain an important, convenient, and frequently used conduit
for surgical revascularization.6 A number of technical advances
have been proposed over the past decade, such as off-pump
CABG and no-touch SV harvesting; nevertheless, the rates of
Introduction
Despite the increased utility of drug-eluting stents, coronary
artery bypass grafting (CABG) remains the treatment of choice
in patients with multi-vessel disease or diabetes due to increased
freedom from recurrent angina, ischaemic events, and need for
repeat intervention.1–3
* Corresponding author. Tel: +44 141 3301977; Fax: +0141 3303360, Email: andrew.h.baker@glasgow.ac.uk
& The Author 2013. Published by Oxford University Press on behalf of the European Society of Cardiology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-
commercial use, distribution, and reproduction in any medium, provided that the original authorship is properly and fully attributed; the Journal, Learned Society and Oxford
University Press are attributed as the original place of publication with correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but
only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@oup.com

Page 2 of 9
R.A. McDonald et al.
vein graft failure remain largely similar.7,8 It is estimated that 20–
50% of patients suffer from stenotic occlusion of the graft within
5 years.9 The high failure rates highlight the need for novel thera-
peutic interventions in the setting of CABG. The principal pro-
cesses responsible for vein graft failure are intimal hyperplasia
[promoted by enhanced vascular smooth muscle cell (SMC) pro-
liferation, migration, and extracellular matrix deposition] and
superimposed atherosclerosis (reviewed in Wan et al.10). Although
aggressive lipid-lowering therapy has delayed vein graft failure in
patients, there is an urgent need for alternative therapies which
can curtail the early pathological remodelling in the short term
in order to improve long-term patency rates.
In vitro models
In vitro experiments were performed using isolated primary human
SV-derived endothelial cells (EC) and SMCs.
Statistical analysis
Data are presented as mean + standard error of the mean (SEM). For
the comparison of mean values, Bartlett’s test for equal variances was
performed—there was no evidence of heterogeneous variances
between groups for any of the comparisons. Visual assessment was
used to check for any lack of normality; as there was no evidence of
this, one-way ANOVA followed by Tukey’s multiple comparison test
(for comparison of more than two groups) or Student’s t-test (for
comparison of two groups) was carried out. For all the quantitative
real-time PCR (qRT-PCR) experiments, values are expressed as fold
change. All statistical analyses were carried out using GraphPad
Prism version 4 (GraphPad Software, USA), other than the microRNA
array data. The microRNA array data were analysed in the DataAs-
sistTM software (Life Technologies). Following manual normalization
identification, miR-199a-3p was chosen as a reference gene to normal-
ize the data, since its standard deviation across all samples was the
lowest for all microRNAs on the chip. One-way ANOVA with Benja-
mini–Hochberg false discovery rate24 (FDR) adjustment for multiple
testing was carried out. We restricted our validation of miRNAs to
those that were up-regulated .4-fold and which were significant at
a 10% FDR in the TaqMan Low Density Array (TLDA) experiment.
For detailed descriptions of materials and methods, see Supplemen-
tary material online.
MicroRNAs belong to a recently discovered class of small, en-
dogenous non-coding RNA molecules that negatively regulate
gene expression by targeting specific messenger RNAs and
induce their degradation or translational repression.11 A number
of reports have highlighted a role for miRNA in SMC proliferation
and migration associated with vascular pathologies (reviewed in
McDonald et al.12). MicroRNA profiling in normal and atheroscler-
otic human arteries demonstrated that miR-21 was up-regulated in
the SMC layer of atherosclerotic vessels.13,14 Furthermore, previ-
ous reports from two independent groups demonstrated that
miR-145 levels were down-regulated following balloon injury and
adenoviral-mediated over-expression of miR-145 reduced intimal
lesion formation.15,16 Torella et al.17 demonstrated that adenoviral
mediated over-expression of miR-133a reduced neointimal forma-
tion and antagomiR to miR-133a exacerbated neointimal formation
following balloon injury. These studies highlight that miRNAs are
dysregulated at the onset of vascular injury, and as a result, modu-
lation of miRNA levels may represent a therapeutic target in a
number of vascular pathologies. However, no studies to date
have assessed the role of miRNAs in the context of neointima for-
mation associated with vein graft failure. Accordingly, we sought to
identify aberrantly expressed miRNAs in models of vein graft
failure. We report using mouse, pig, and human models that a
number of miRNAs were dysregulated during vein graft remodel-
ling. We also highlight that genetic ablation of miR-21 substantially
limited neointima formation in mouse vein grafts in vivo. To inves-
tigate the translational application of these findings, human saphe-
nous veins (HSVs) were exposed to anti-miR-21 ex vivo. In this
model, we show that de-repression of several target genes is
achieved and a significant reduction in neointima size is observed,
suggesting that localized inhibition of miR-21 may provide a novel
therapeutic approach to prevent vein graft neointima formation.
Results
Global microRNA profiling in ungrafted
and grafted saphenous veins
In order to identify aberrantly expressed miRNA during neointima for-
mation in the setting of vein graft failure, we compared miRNA expres-
sion patterns by microarray analysis in ungrafted and grafted porcine
SVs isolated 7 and 28 days post-engraftment (Figure 1A). Global
miRNA profiling analysis revealed that 21 out of 377 miRNAs were
up-regulated following engraftment (see Supplementary material
online, Figure S1 and Table S1), 6 of which were up-regulated more
than 4-fold (FDR , 0.1) at 7 and 28 days post-engraftment
(Figure 1B). From this data set, we focused on miR-21 due to the sub-
stantial up-regulation observed in pig grafts compared with controls
(Figure 1C) and since it has previously been implicated in SMC and
fibroblast proliferation following acute vascular injury.25,26
miR-21 expression is elevated in multiple
models of vein graft disease
In order to validate and further quantify the expression levels of
miR-21 during the progression of vein graft neointimal formation,
we profiled miR-21 levels in three well-characterized models of
vein graft disease, using qRT-PCR analysis. These models were (i)
in vivo porcine model of interposition grafting, (ii) the interposition
mouse vein graft model (jugular vein into the right common
femoral artery), and (iii) the ex vivo model of HSV neointimal for-
mation using surplus vein tissue harvested at the time of CABG. In
the porcine model, qRT-PCR confirmed that miR-21 levels were
elevated at 7 days following grafting and remained elevated at 28
days (Figure 1D). We observed an ꢀ7-fold increase in levels at
Methods
Vein graft models
Several established models of vein grafting were utilized in this study.
We utilized two mouse models of vein grafting; interpositional graft-
ing18 was used for miR-21 profiling, and the isogenic mouse vein
graft model19 was used for all other experiments. Porcine SV-carotid
interpositional grafting20 and ex vivo culture of human SV segments21
were also used. In addition, we assessed failed grafts removed from
patients at least 5 years post-CABG. miR-21 knockout mice were pre-
viously described.22,23

Dysregulation of miRNA-21 in response to vein grafting
Page 3 of 9
Figure 1 Global microRNA profiling and assessment of miR-21 in vein grafting. (A) A schematic diagram illustrating how the samples were
prepared for global microRNA expression, using microfluidic cards. Ungrafted saphenous veins were compared with veins subjected to vein
grafting for a period of 7 and 28 days. We evaluated only targets which were .4-fold up-regulated at both time points. (B) A heat map of
the only microRNAs which were significantly up-regulated .4-fold; the table summarizes the fold change, P-values, and the false discovery
rate (FDR) for each microRNA. (C) Relative quantification of the miR-21 array results normalized to miR-199a-3p. (D) Verification of
TLDA array by real-time-PCR, demonstrating up-regulation of miR-21 in porcine veins following grafting for 7 and 28 days (n ¼ 6/group).
(E) Expression of miR-21 in control ungrafted jugular vein (JV) and vein graft (VG) from mice (n ¼ 5/group). (F) Real-time PCR for miR-21
expression in HSV subjected to culture for a period of 0, 7, 14, or 28 days to allow neointimal lesions to develop (n ¼ 6/group). Error bars
denote SEM; *P , 0.05, **P , 0.01. Data were analysed by one-way ANOVA with a Tukey’s post-test.
both time points compared with ungrafted SV (P , 0.01, n ¼ 6/
group), which is consistent with the changes observed in our
TLDA analysis (Figure 1C). This elevation in miR-21 levels was par-
alleled in the mouse vein graft model, with miR-21 levels elevated
in grafts at 28 days compared with ungrafted conduits (jugular
veins) (P , 0.001, n ¼ 4–5, Figure 1E). Finally, we assessed levels
in HSV ex vivo organ cultures. miR-21 was significantly up-regulated
2.5-fold in vein segments cultured for 7 and 14 days (P , 0.05, n ¼ 6,
Figure 1F). Therefore, in three independent models representative of
vein graft neointima formation, we observed that miR-21 levels were
significantly elevated when analysed by qRT-PCR.
miR-21. miR-21 expression was low in porcine carotid arteries
and undetectable in ungrafted SVs (Figure 2A and B). However,
we observed positive staining for miR-21 in porcine vein grafts,
in the adventitial, medial, and neointimal layers at both 7 and 28
days (Figure 2C and D). We performed immunohistochemistry
with a-actin on serial sections since SMCs are integral to the for-
mation of neointimal lesions. In vein grafts, we noted that miR-21
was located in all three layers of the vessel wall, in regions of the
graft expressing a-actin (Figure 2K and L).
In a second mouse model of vein grafting, the isogenic graft
model (vena cava into the right common carotid artery), miR-21
levels were undetectable in the ungrafted inferior vena cava
and low in the carotid artery compared with the wide spread
and high-level expression in grafted tissue (Figure 3). We per-
formed immunohistochemistry for a-actin and mac-2 to determine
whether the cells expressing miR-21 were SMCs or macrophages,
Localization of miR-21 in models of vein
graft neointimal formation
In order to ascertain the localization of miR-21 in control vessels
and veins post-grafting, we performed in situ hybridization for

Page 4 of 9
R.A. McDonald et al.
Figure 2 Localization of miR-21 at 7 and 28 days post-engraftment in a porcine model. (A–D) In situ hybridization (violet) of miR-21 in the
carotid artery, ungrafted saphenous vein, and grafted saphenous vein at 7 and 28 days post-engraftment, respectively. (E–H) In situ hybridization
on the same sections with a scrambled probe. (I–L and M–P) SMA and control IgG staining, respectively, in serial sections from the same vein
grafts. Arrows indicate the neointimal layer; scale bar represents 100 mm.
Figure 3 Localization of miR-21 in mouse vessels. In situ localization of miR-21 in the mouse inferior vena cava (A), carotid artery (B), and vein
grafts harvested 28 days post-engraftment at high (C) and low magnification (D). (E–H) The corresponding control in situ hybridization with a
scrambled probe. Filled arrows indicate neointimal thickness; scale bar represents 200 mm, applicable to all panels.
respectively. Comparison of the staining pattern for a-actin, mac-2,
and proliferating cell nuclear antigen (PCNA) revealed that miR-21
is expressed in regions of the graft which stained positive for both
SMC actin (a-actin) and macrophages (mac-2), although not all
a-actin-positive cells expressed miR-21 (Figure 4A–D). These
studies on sequential serial sections suggest that miR-21 is
expressed in proliferating SMCs in the neointima and macrophages
and actin-positive cells in the adventitial layer of the vein grafts. We

Dysregulation of miRNA-21 in response to vein grafting
Page 5 of 9
Figure 4 Immunolocalization of cells expressing miR-21 in murine vein grafts. (A) In situ hybridization with a scrambled LNA probe. (B) In situ
hybridization for miR-21 in a mouse vein graft at 28 days. (C–F) Immunohistochemistry staining for proliferating cell nuclear antigens, smooth
muscle cells (SMA), macrophages (mac-2), and control IgG, respectively. Scale bar represents 100 mm.
Figure 5 miR-21 expression in human vein subjected to culture. (A–D) In situ hybridization of miR-21 in human saphenous veins following 0,
4, 7, and 14 days of cell culture. (E–H) In situ hybridization on serial sections with a scrambled probe. (I–L) Serial sections stained with
anti-a-actin antibodies (SMA) to determine smooth muscle cells. Scale bar represents 100 mm, applicable to all panels.
next performed immunohistochemistry for the fibroblast markers
vimentin and FSP-1.27 Cells staining positive for vimentin and FSP-1
were found extensively in the adventitia and a large proportion of
cells in the neointimal layer, suggesting that myofibroblasts also
contribute to miR-21 expression in the neointimal layer in this
mouse model (see Supplementary material online, Figure S2).
However, further detailed co-localization studies are needed to
definitively demonstrate the cell types responsible for miR-21
expression.
In situ hybridization for miR-21 in surgically prepared HSV seg-
ments demonstrated that miR-21 was expressed in medial and
neointimal SMCs (Figure 5). Immunohistochemistry in sequential

Page 6 of 9
R.A. McDonald et al.
serial sections demonstrated that miR-21 expression was localized
in areas which stained positive for a-actin (Figure 5J–L).
Thus, in concordance with the qRT-PCR analysis, we observed
elevated levels of miR-21 in all three models of vein grafting,
with the expression localized to multiple regions of the graft.
engrafted into wild-type mice. Neointimal size was significantly
reduced compared with wild-type grafts in wild-type mice
(Figure 6A), suggesting that miR-21 expression in the engrafted
vessel is critical to neointima formation.
Pharmacological knockdown of miR-21
expression in cultured HSV
Effect of genetic loss of miR-21 on
neointimal formation in vein grafts
To investigate the translational application of our findings and
evaluate whether it is possible to achieve pharmacological knock-
down of miR-21 in HSVs, the ex vivo culture model was utilized. Cul-
turing HSV in the presence of anti-miR-21 for 7 or 14 days resulted
in a .95% knockdown in miR-21 expression (Figure 7A). The ex-
pression of previously identified miR-21 target genes25,29–31 was
analysed by qRT-PCR. Compared with anti-miR-ctl-treated HSVs,
anti-miR-21 treatment caused significant de-repression of STAT3,
PTEN, and BMPR2 at 14 days (Figure 7B). However, PDCD-4 and
TIMP3 expression were not significantly altered (Figure 7B). Neoin-
tima formation was significantly reduced in anti-miR-21-treated
vessels (Figure 7C and D).
In order to address whether the elevation of miR-21 plays an im-
portant role in the development of vein graft neointimal formation,
we performed isogenic vein grafting in miR-21 knockout mice and
wild-type controls. At 28 days post-engraftment, the neointimal
area was dramatically reduced by 81% in miR-21 knockout mice
compared with wild-type controls (P , 0.001, n ¼ 7–10/group)
(Figure 6A and B). Additionally, neointimal lesions in miR-21 knock-
out mice had significantly lower SMC content than controls (see
Supplementary material online, Figure S3).
In this mouse model, it has previously been demonstrated that
cells from both the donor vessel and recipient contribute to the
progression of neointima formation.28 To determine whether abla-
tion of miR-21 in the donor vessel is sufficient to prevent neoin-
tima formation, donor veins from miR-21 knockout mice were
Owing to the limited availability of intact human SVs, we per-
formed further experiments with anti-miR-control and anti-miR-21
in primary SV-derived EC and SMCs. In these experiments, we
Figure 6 Effect of miR-21 ablation on neointimal formation in a mouse model of vein grafting. (A) The neointimal area 28 days post-grafting in
wild-type mice, miR-21 knockouts, and miR-21 knockout veins engrafted into wild-type mice. (B) Vessel wall thickness in grafts from wild-type
and knockout mice at 0 and 28 days; sections are stained with elastin van Gieson. Arrows indicate the neointimal layer. n ¼ 5–10, ***P , 0.001
vs. wild-type. Scale bar represents 200 mm, applicable to all panels.

Dysregulation of miRNA-21 in response to vein grafting
Page 7 of 9
Figure 7 Effect of pharmacological knockdown of miR-21 in HSVs and miR-21 expression in failed human vein grafts. HSV segments were
cultured for 7 and 14 days in the presence of 5000 nM anti-miR-ctl or anti-miR-21 (n ¼ 5/group), then RNA was isolated from the vessels, and
expression was measured by quantitative real-time PCR. (A) miR-21 expression. (B) Expression of putative miR-21 target genes. (C) Neointimal
thickness. (D) Representative images of elastic van Gieson-stained sections from Day 14 samples. (E) In situ hybridization with a scrambled
microRNA probe, miR-21 probe, and immunohistochemistry for smooth muscle cell actin in a failed human vein graft. Scale bar represents
100 mm, applicable to all panels. *P , 0.05 vs. anti-miR-ctl; **P , 0.01 vs. anti-miR-ctl, ***P , 0.001 vs. anti-miR-ctl, ##P , 0.01 vs. Day 0,
###P , 0.001 vs. Day 0.
aimed to identify any off-target effects of the anti-miR-control and
anti-miR-21 treatments; hence, we transfected these cells with anti-
miRs at a range of doses and analysed miR-21 expression compared
with mock-transfected cells 72 h post-transfection. These data
demonstrated that anti-miR-21, but not anti-miR-control transfec-
tion produced a substantial knockdown of miR-21 levels (see Sup-
plementary material online, Figure S4A and B). We also noted that
there were small but sometimes significant variations in miR-21
levels in anti-miR-control samples, but these are small and are un-
likely to impact experimental outcome (see Supplementary material
online, Figure S4A and B). Taken together, these results suggest that
anti-miR-21 treatment is a viable strategy to knockdown miR-21
levels in venous SMC pre-engraftment.
stain positive for SMC-actin (see Figure 7E and Supplementary ma-
terial online, Figure S5).
Discussion
This study is the first to document miRNA dysregulation in the
context of vein graft neointima formation. It is also the first to de-
monstrate a functional role for miR-21 in neointimal formation
following vein grafting. Our miRNA profiling identified 21
miRNAs which were significantly up-regulated in porcine vein
grafts 7 and 28 days post-engraftment. We validated this sustained
up-regulation of miR-21 expression in these porcine vein grafts and
relevant murine and human models of vein graft neointimal forma-
tion by qRT-PCR. Our in situ hybridization studies demonstrated
that miR-21 was abundantly expressed in the neointimal layer of
the venous wall following engraftment in porcine and murine
models and failed human grafts. Furthermore, our ex vivo HSV
model confirmed this up-regulation of miR-21 levels in cells within
the neointimal layer. These studies suggested that miR-21 might
play a pathological role during neointimal formation in the setting
of vein graft failure; hence, we performed vein grafting in miR-21-
ablated mice. The absence of miR-21 in these mice substantially
miR-21 expression in failed human vein
grafts
In order to demonstrate the importance of miR-21 in the clinical
setting of vein graft failure, we performed in situ hybridization for
miR-21 in failed human vein grafts. The staining pattern in these
veins is very similar to that seen in the mouse and porcine vein
grafts, with miR-21 expressed in regions of the graft which also

Page 8 of 9
R.A. McDonald et al.
attenuated neointimal formation and SMC accumulation in intimal
lesions. Furthermore, engraftment of veins from miR-21 mice into
wild-type mice resulted in the attenuation of neointima formation,
suggesting that miR-21 expression in the engrafted vessel is critical
to neointimal development. This has important implications for
the development of miR-21 therapeutics, as it suggests that localized
treatment to knockdown miR-21 expression in the vein may be suf-
ficient for therapeutic efficacy.
In HSV samples treated with anti-miR-21, we analysed changes
in gene expression of several previously proposed targets of
miR-21. PTEN is an established target known to be expressed in
fibroblasts, EC, and SMCs, where it regulates cell survival/apoptosis
and is thought to have a key role in many cardiovascular diseases.32
In agreement with previous studies,14,26,33 we demonstrated an
increase in PTEN expression in treated vessels. We also found
significant de-repression of STAT3 and BMPR2, which have been
shown to be direct targets of miR-21 in mesenchymal stem
cells31 and pulmonary vascular SMCs,34 respectively. In wild-type
mice, we found miR-21 expression localized to regions of the
neointima and adventitia where PCNAs, SMCs, and fibroblast
markers are also expressed. This suggests that the increase in
miR-21 expression may contribute to the progression of neointima
formation by promoting SMC and fibroblast proliferation and
cell survival.
This is consistent with previous reports showing that knock-
down of miR-21 levels in arterial SMCs and fibroblasts reduced
rates of proliferation, migration, and neointimal formation in a
rat balloon injury model, which was at least in part caused by de-
repression of PTEN and BCL-2.14,25,26 A subsequent study in a
mouse model of abdominal aneurysm demonstrated that lentiviral-
mediated over-expression of miR-21 reduced PTEN expression
and increased SMC proliferation.33 In the setting of vascular injury,
these studies are important since over-expression and gene knock-
out studies demonstrate a role for PTEN in SMC proliferation and
neointimal formation.35,36 Moreover, previous studies suggested
that PDCD-4 is down-regulated in the rat model of balloon injury,
and over-expression with adenoviral vectors increased apoptosis
and reduced SMC proliferation;30 however, we did not find any sig-
nificant change in PDCD-4 levels in our ex vivo HSV studies. There
are also other reported targets of miR-21 which could play a role in
vein graft failure. Recently, a study by Wang et al.14 in atherosclerotic
arteries demonstrated that miR-21 targeted tropomyosin-1, a
protein implicated in the formation, stabilization, and regulation of
cytoskeletal actin filaments. A change in tropomyosin may play a
role in the reduced neointimal formation seen in the miR-21 knock-
out mouse vein graft study presented here, possibly via a reduction
in SMC migration. Further detailed mechanistic studies are required
to address the role of these targets in vein graft pathophysiology.
To investigate the translational application of our findings, we
performed a pilot study to investigate the potential of knocking
down miR-21 expression in intact human SVs. Although this ex vivo
model has some limitations, such as the development of smaller
neointimal lesions than those seen in vivo due to the lack of flow
(shear stress) and infiltrating inflammatory cells, which contribute
to neointimal formation in vivo, this model allows researchers to
focus on the role of matrix remodelling, SMC proliferation, and mi-
gration. Using this model, we have demonstrated that it is possible to
manipulate miRNA expression and observe changes in target gene
expression in clinically relevant tissue samples.
Therapeutic potential
CABG provides an opportunity for the delivery of agents that can
modulate the pathophysiology of vein graft neointima formation. In
this study, we suggest that manipulation of miRNA may be one
such therapeutic strategy. The manipulation of miR-21 levels in
the venous wall at the time of engraftment through ex vivo treat-
ment of the harvested vein could lead to localized manipulation
of miRNA. This would negate the need for systemic delivery,
which may result in the knockdown of miR-21 in other tissues, po-
tentially creating off-target safety issues. We have demonstrated
that anti-miRs can be used to knockdown miR-expression in
HSV and that the level of inhibition achieved was sufficient to
mediate target de-repression and reduce neointima formation.
However, owing to the short exposure time available for ex vivo
manipulation of the vein in the clinical setting (up to 30 min), it
will be necessary to develop more efficient methods of inhibiting
miR-21 in intact veins. A highly efficient delivery system is required
to provide rapid and efficient uptake into the venous wall. Previous
pre-clinical studies which have utilized decoy oligonucleotides
directed against E2F showed efficient manipulation of the target
and therapeutic efficacy when delivered to the vein under pres-
sure,37,38 although clinical trials failed to show efficacy.39 It is well
documented that viral vectors, specifically adenovirus, are efficient
vein graft delivery vectors;40 hence, the design of viral approaches
for efficient delivery is a further option.
In summary, our study demonstrates that a number of miRNAs
are modulated by the process of vein grafting and that miR-21 is
significantly up-regulated in the neointimal layer of veins following
engraftment. Further, genetic deletion of miR-21 substantially
reduced neointimal formation and SMC accumulation within
these lesions. This suggests that modulation of miR-21 level has
therapeutic potential in the setting of vein graft failure.
Supplementary material
Supplementary material is available at European Heart Journal
online.
Acknowledgements
The authors thank Nicola Britton and Gregor Aitchison for tech-
nical assistance and Professor Eric Olson (University of Texas
Southwestern Medical Center) for the miR-21 knockout mice.
Funding
This work was funded by the British Heart Foundation (programme
grant no. RG/09/005/27915). A.H.B. is supported by the British
Heart Foundation Chair of Translational Cardiovascular Sciences.
Conflict of interest: E.v.R. is co-founder and former employee of
MiRagen Therapeutics. She is now Associate Professor at Hubrecht
Institute, University Medical Center Utrecht.
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