Accelerated intimal hyperplasia in aortocoronary internal mammary vein grafts in minipigs.

Abstract

More than 50% of aortocoronary saphenous vein grafts are occluded 10 years after surgery. Intimal hyperplasia is the initial critical step in the progression toward occlusion. Internal mammary veins, which are physiologically prone to less hydrostatic pressure, may undergo an accelerated progression to intimal hyperplasia and thus be suitable for investigation of the mechanisms of aortocoronary vein graft disease.
Six minipigs underwent aortocoronary bypass grafting using standard cardiopulmonary bypass and cardioplegic arrest. Mammary vein were grafted in a reversed manner from ascending aorta to left anterior descending coronary artery (LAD). The proximal LAD was ligated, rendering the anterior left ventricle vein graft-dependent. Minipigs were killed after 4 weeks, and vein grafts were harvested. Histological and immunohistological investigation were performed with respect to morphometric analysis, endothelial damage/dysfunction (v-Willebrand-factor (vWF)), smooth muscle cells (alpha-smooth actin) and proliferation rate (proliferation marker Ki 67).
Mean intimal area of vein grafts was increased compared to ungrafted mammary veins. Intimal hyperplasia in vein grafts was characterized by massive accumulation of smooth muscle cells with a high proliferation rate and endothelial perturbation. Significant (p = 0.001) intimal hyperplasia of the grafted mammary vein compared to the ungrafted mammary vein was found. These changes were absent in ungrafted mammary veins.
The present study demonstrates a pig model of aortocoronary vein graft intimal hyperplasia which is characterized by an accelerated progression within internal mammary veins. The model is suitable to investigate the pathophysiology of aortocoronary vein graft intimal hyperplasia as well as therapeutic approaches.

Full text

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BioMed Central
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Journal of Cardiothoracic Surgery
Open Access
Research article
Accelerated intimal hyperplasia in aortocoronary internal
mammary vein grafts in minipigs
Aron Frederik Popov*1, Hilmar Dorge1, Jose Hinz2, Jan Dieter Schmitto1,
Tomislav Stojanovic1, Ralf Seipelt1, Vassilios Didilis3 and
Friedrich Albert Schoendube1
Address: 1Department of Thoracic Cardiovascular Surgery, University of Göttingen, Germany, 2Department of Anaesthesiology, Emergency and
Intensive Care Medicine, University of Göttingen, Germany and 3Department of Cardiothoracic surgery, Democritus University of Thrace, Greece
Email: Aron Frederik Popov* - Popov@med.uni-goettingen.de; Hilmar Dorge - doerge@med.uni-goettingen.de;
Jose Hinz - mailto@josehinz.de; Jan Dieter Schmitto - Schmitto@med.uni-goettingen.de; Tomislav Stojanovic - tstojan@gwdg.de;
Ralf Seipelt - rseipelt@med.uni-goettingen.de; Vassilios Didilis - vdidilis@med.duth.gr; Friedrich Albert Schoendube - Schondube@med.uni-
goettingen.de
* Corresponding author
Abstract
Background: More than 50% of aortocoronary saphenous vein grafts are occluded 10 years after
surgery. Intimal hyperplasia is the initial critical step in the progression toward occlusion. Internal
mammary veins, which are physiologically prone to less hydrostatic pressure, may undergo an
accelerated progression to intimal hyperplasia and thus be suitable for investigation of the
mechanisms of aortocoronary vein graft disease.
Methods: Six minipigs underwent aortocoronary bypass grafting using standard cardiopulmonary
bypass and cardioplegic arrest. Mammary vein were grafted in a reversed manner from ascending
aorta to left anterior descending coronary artery (LAD). The proximal LAD was ligated, rendering
the anterior left ventricle vein graft-dependent. Minipigs were killed after 4 weeks, and vein grafts
were harvested. Histological and immunohistological investigation were performed with respect to
morphometric analysis, endothelial damage/dysfunction (v-Willebrand-factor (vWF)), smooth
muscle cells (α-smooth actin) and proliferation rate (proliferation marker Ki 67).
Results: Mean intimal area of vein grafts was increased compared to ungrafted mammary veins.
Intimal hyperplasia in vein grafts was characterized by massive accumulation of smooth muscle cells
with a high proliferation rate and endothelial perturbation. Significant (p = 0.001) intimal hyperplasia
of the grafted mammary vein compared to the ungrafted mammary vein was found. These changes
were absent in ungrafted mammary veins.
Conclusion: The present study demonstrates a pig model of aortocoronary vein graft intimal
hyperplasia which is characterized by an accelerated progression within internal mammary veins.
The model is suitable to investigate the pathophysiology of aortocoronary vein graft intimal
hyperplasia as well as therapeutic approaches.
Published: 29 April 2008
Journal of Cardiothoracic Surgery 2008, 3:20doi:10.1186/1749-8090-3-20
Received: 26 December 2007
Accepted: 29 April 2008
This article is available from: http://www.cardiothoracicsurgery.org/content/3/1/20
© 2008 Popov et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Introduction
The saphenous vein is still a conduit of choice for coro-
nary artery bypass grafting. Following arterializations,
vein grafts undergo immediate injury like ischemia and
wall stress. The histological changes associated with vein
graft failure are defined as intimal hyperplasia. They
include acute thrombosis or early medial and intimal
thickening that may be focally progressive. Further reason
is late artheroma formation, which is the most important
cause of failure beyond five years after implantation [1].
This vein graft failure is result of progressive thickening of
the intima and media acting over the first months and
slow process of atherosclerosis over years [2]. According
to this, major limitation for its use is the high graft occlu-
sion rate, which increase from 8% early on 14th day, to
13% at 1 year up to 20% at five years after operation [3].
Vein graft failure and intimal hyperplasia have already
been shown in large animals, like dogs and pigs [4-8]. The
advantage of large animal is the more human like wall of
the vein graft compared with veins of small animal.
Because of their size breeds of 50 kilogram are sufficient
for standard arthrosclerosis long term research. However,
these breeds can gain up to 1 kilogram per day and reach
weights of more than 200 kilogram and are difficult to
handle under laboratory conditions. The anatomical
structures of these pigs are not suited for standard inter-
ventional cardiac techniques, and equipment used for
human procedures can not be easily applied. After six
month the Goettingen minipig reached a body weight of
50 kilogram and gain up no more. The anatomical condi-
tions like size, structure and functions of the heart of the
Goettingen minipig approach those of humans allowing a
transfer of the animal results obtained. The size of veins
and coronary artery in these pigs are ideal for coronary
artery surgery [9].
Because of the great differences in hydrostatic pressure
acing along the blood vessels in erect posture, saphenous
vein is exposed to greater transmural pressures than inter-
nal mammaria vein. Internal mammaria vein may there-
fore suitable for an accelerated model of intimal
hyperplasia and atherosclerosis.
In the present study we used a minipig model and internal
mammary vein for aortocoronary bypass surgery. Internal
mammaria vein were grafted in reversed manner from the
ascending aorta to the left anterior descending coronary
artery followed from ligating the proximal left anterior
descending coronary. Our objective was to establish a
model for accelerated intimal hyperplasia within four
weeks in aortocoronary internal mammary vein grafts in
minipig. Furthermore this model should permit investiga-
tion of the pathophysiology of intimal hyperplasia and
assessment of pharmacological strategies for its preven-
tion.
Methods
Six female Goettingen minipigs pigs (weight 53.4 ± 3 kg,
age 10–12 month) underwent aortocoronary bypass graft-
ing with mammary vein as a free transplant with cardiop-
ulmonary bypass. Minipigs were killed after four weeks,
and vein grafts were harvested. Histological and immuno-
histological investigation were performed with respect to
morphometric analysis, endothelial damage/dysfunction,
smooth muscle cells and proliferation rate. The study was
approved in accordance with the ethic guidelines of the
local ethics committee and federal laws. All minipigs
received human care in accordance with the "Guide for
the Care and Use of Laboratory Animals" as revised by the
National Institutes of Health in 1985.
Anesthesia
On the day of surgery all pigs received an intramuscular
injection of 10 mg/kg azaperone (Stressnil®, Janssen,
Neuss, Germany). Pigs were anesthetized and intubated
with a combination of ketamine 10 mg/kg and xylazine
0.1 mg/kg by an intravenous injection and a prophylactic
dose of antibiotic dose with 2 mg/kg cefquinome
(Cobactan®, Intervet Deutschland GmbH, Untersch-
leißheim, Germany) were given.
After induction and intubation, the pigs were ventilated
mechanically using a mixture of isoflurane 1.5–3 Vol%,
and pure oxygen. Anaesthesia was maintained with piritr-
amid 75 μg/kg/h (Dipidolor®, Janssen, Neuss, Germany)
and ketamine (Ketanest®, Deltaselect, Dreieich, Germany)
5–8 mg/kg/h. Intravenous line was inserted in an ear vein
and arterial line were inserted and in the femoral artery for
continuous blood pressure monitoring.
Operative procedure
The pigs were placed in a dorsal recumbence, and a
median sternotomy from the tip of the xyphoid bone to
the suprasternal notch was performed. Following place-
ment of a sternal retractor, the left internal mammary vein
was dissected free of surrounding tissue by "no touch"
technique. The dissection was begun distally at the artery's
bifurcation and proceeded cephaladly as far as possible
into the chest cavity apex. From the distal end of the vein,
a short segment was separated for further histologic and
immunohistochemistry investigations.
The pericardium was opened longitudinally. Inactivation
of the coagulation system necessary for the cardiopulmo-
nary bypass was achieved by an injection of 400 IU
heparin/kg (Liquemin®, 25000, Hoffmann-La Roche,
Grenach-Wyhlen, Germany). The cardiopulmonary
bypass was established via cannulation of the ascending

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aorta and right atrium. The body temperature was cooled
to 32°C under extracorporeal circulation. Cardioplegic
cardiac arrest was achieved by antegrade infusion of cold
crystalloid cardiplegia (Bretschneider HTK Custodiol®,
Köhler Chemie, Alsbad, Germany). Following cardiac
arrest the left anterior descending coronary artery was pre-
pared for. After preparing the left anterior descending cor-
onary artery, a suited location was chosen. The internal
mammary vein was connected as free graft to the artery
using conventional suture technique (7/0 Prolene®,
Ethicon, Inc., Somerville, NJ, USA). After connection of
the distal anastomoses, minipigs were slowly rewarmed to
36°C. The proximal anastomoses were performed during
tangential clamping of the ascending aorta while reper-
fusion of the beating heart on cardiopulmonary bypass.
After clamp removal, the proximal left anterior descend-
ing was ligated with a suture (5/0 Prolene®, Ethicon, Inc.,
Somerville, NJ, USA) to guarantee exclusive bypass flow.
Sufficient blood flow within the bypass was checked by
palpation of arterial pulsation and the absence of macro-
scopic sudden myocardial infarction. After coming off
bypass protamine was adminstrated (Protamin®, Hoff-
mann-La Roche, Grenach-Wyhlen, Germany) to neutral-
ize the heparin dosage to 100%. Temporary substernal
and intrapleural drainage tubes (28 mm, Sherwood Med-
ical, Crawley, England) were placed and connected to
continuous suction system (Redovac®, Sherwood Medical,
Crawley, England). The chest was closed using stainless
steel wires (1.75 mm, Deknatel®, Lübeck, Germany). With
the beginning of skin suture (2/0 Prolene®, Ethicon, Inc.,
Somerville, NJ, USA) the inhalation mixture was changed
to pure oxygen and the respiratory minute volume was
slowly reduced until spontaneous respiration was present.
General anesthesia was terminated and the minipigs were
extubated upon resumption of vagal reflexes. Postopera-
tive analgesic treatment was not performed. Before extu-
bation the thoracic drainages were removed.
Four weeks later the minipigs were anaesthetized again in
the way describe above and the chest was re-opened. After
macroscopic documentation concerning the function of
the mammary vein graft and examined of signs of infec-
tion, the minipigs were killed with in injection of potas-
sium (Kaliumchlorid 7.45%,
Germany) under anesthesia. The complete mammary vein
bypass was carefully removed and perfusion-fixed 4% for-
maldehyde solution. The bypass was infused with fixative
for 10 minutes at a pressure of 80 mmHg according to a
standard protocol [10].
Braun, Melsungen,
Histological techniques
Morphometric analysis
The diameters of the harvested grafts were measured close
to the proximal anastomosis. This part of the graft was
adjacent to the histological sample of the native vein.
Perfusion-fixed vein segments were embedded in paraffin,
and cut transversely into 5 μm thick sections, which were
stained in haematoxylin-eosin and van Giesson stain to
make the elastic fibers visible. Segments of internal mam-
mary vein grafts and ungrafted internal mammary vein
wall dimensions were measured by computer-aided plan-
imetry [Hardware: Olympus® BH-2 microscope video
camera, head (JVC TK-870E), computer (Victor V386A,
Victor Technologies); software: SoftaOlympus DP-Soft
3.1® image-analysis system (Olympus Europe Holding
GmbH, Hamburg, Germany)]. Wall thickness of intima
was measured from the subendothelial basal lamina to
the internal elastic lamina, from where media extends to
the adventitial layer in four regions of interests with
respect of the cardinal points of a compass (North, East,
South, West). Luminal areas were also determined from
these histological slices.
Immunohistochemistry and cell proliferation analysis
Proliferation of the intimal cells was assessed using
immunhistochemical reaction with monoclonal antigen
[11,12] (Ki-67, Dako GmbH, Hamburg, Germany).
Smooth muscle cells proliferation throughout the new
intima were identified by using monoclonal antibodies
directed against α-smooth-muscle-actin (clone 1A4, Dako
GmbH, Hamburg, Germany). Vein sections were also
stained with rabbit anti-human von Willebrand human
factor (vWF, Dako GmbH, Hamburg, Germany) to iden-
tify dysfunction of endothelial cells.
Statistical analysis
All date were tested for normal distribution with the Kol-
mogorov-Smirnov-test and then analyzed further with
parametric methods as indicated using commercial statis-
tic software (Statistica 5.1.®, StatSoft Inc., Tulsa, OK, USA).
Comparison between ungrafted vein and graft-vein was
carried out with a Mann-Whitney U-Test. Results were
considered significant if p was less than 0.05.
Results
Eight animals entered into the protocol, six were used in
the final analysis. The initial two animals developed
intractable ventricular fibrillation during coronary occlu-
sion stage or immediately afterwards and it was impossi-
ble to defibrillate the heart using electric shock. There
were killed, humanely. In the final analysis no animal
died prematurely. None developed incision infection.
The native veins were investigated after harvesting imme-
diately. Grafts of the mammaria vein were investigated
four weeks after coronary artery bypass grafting. None of
the studied minipigs showed cardiac events (i.e. infarc-
tion, arrhythmic, sudden death). Luminal area differed
not between native vein (1.50–12.07(4.25) mm2) and
graft (0.03–26.08(0.96) mm2).

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Histological section demonstrated hyperplasia of new
intima. Immunohistochemistry showed a proliferation of
the intimal cells. This was confirmed by massive accumu-
lation of smooth muscle cells with a high proliferation
rate and endothelial perturbation and an increase of von
Willebrand factor as an indictor of endothelial dysfunc-
tion (Figure 1). Significant intimal hyperplasia of the
grafted vein compared to the native vein was found by an
increase of wall thickness in the four regions of interests
(Table 1).
Discussion
The intent of this study was to describe a new animal
model with respect to accelerated intimal hyperplasia in
aortocoronary vein grafts. Intimal hyperplasia is one of
the dominant mechanisms underlying the occlusion of
veins used in coronary artery bypass surgery. It is driven by
cell proliferation and migration, and elaboration of extra-
cellular matrix, predisposes to atheromatous thickening
of the venous graft, ulceration of the endothelium and
thrombosis [2]. These mechanisms contribute to the high
incidence of occlusion of vein grafts used in coronary
artery bypass surgery.
Histological appearance of an ungrafted internal mammaria vein (A), a representative grafted mammaria vein after four weeks, with measurements of intimal hyperplasia in respect of the cardinal points of a compass (B), proliferating intimal cells in brown (proliferationsmarker KI 67) (C), dysfunction of endothelial cells (vWF) (D)
Figure 1
Histological appearance of an ungrafted internal mammaria vein (A), a representative grafted mammaria vein after four weeks,
with measurements of intimal hyperplasia in respect of the cardinal points of a compass (B), proliferating intimal cells in brown
(proliferationsmarker KI 67) (C), dysfunction of endothelial cells (vWF) (D).

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In our study we chose internal mammary vein grafted as a
free transplant to ascending aorta to left anterior descend-
ing coronary artery. This operative procedure corresponds
with routine aortocoronary bypass surgery with veins in
humans. As the diameter of the used internal mammary
vein is more likely comparable to the diameter of left ante-
rior descending artery, it simplifies aortocoronary bypass
surgery. We found an accelerated intimal hyperplasia in
vein grafts after four weeks indicated by morphometric
analysis, endothelial damage/dysfunction markers, and
proliferation of smooth muscle cells. Smooth muscle cell
proliferation is a key event in the development of athero-
sclerosis, restenosis and intimal hyperplasia [13,14].
This is in accord with theoretical predictions and the
experimental work of others who investigated intima
hyperplasia in animal models. Numerous studies have
documented intimal hyperplasia in vein grafts in small
and large animals [4-8,15].
In mice vena cava were inserted into carotid artery
[15,16]. In rats the ileolumbar vein and inferior vena cava
were placed into abdominal aorta and superficial epigas-
tric vein into femoral artery [17,18]. The jugular vein was
inserted into carotid artery in rabbits [19,20]. In canines
jugular vein were inserted into carotid artery, saphenous
vein were grafted into coronary artery, femoral vein and
jugular vein were placed in femoral artery [8,21-23].
Numerous pig models are described for saphenous vein or
jugular vein interposition into carotid artery [4-7].
However all of these models do not investigate intimal
hyperplasia during aortocoronary bypass surgery. The
present model complements other in vivo models of
venous intimal hyperplasia.
The advantage of our model is the more human like wall
thickness of the vein graft compared with thin wall of
small animal veins. The anastomosis of venous segments
directly into the arterial circulation in small animals rep-
resents a technically demanding model, requiring micro-
surgical expertise [24,25].
We chose a minipig model to show a model for cardiovas-
cular research in respect of intimal hyperplasia in mam-
maria vein grafts. Pigs are one of the readily available
species in which cardiovascular anatomy and physiology
resembles humans. There are many similarities between
the species. In humans and pigs, size and distribution of
coronary arteries are similar [26] with blood flow through
the right and left anterior descending coronary arteries
supplying nearly 80% of the myocardium in pigs. Stand-
ard operative cardiac techniques can be applied in the
minipig using the same equipment used for human pro-
cedures. Size of the vessels and heart of minipig make
them ideal for developing and testing intravascular or
extravascular device prototypes similar in size and config-
uration to the model that may be used in humans. There-
fore, pigs are widely used as a model of intimal
hyperplasia, and they have become the model of choice
for studies in cardiac interventional procedures.
No pig model have already shown vein graft disease of
internal mammary vein which were grafted in a reversed
manner from the ascending aorta to the left anterior
descending coronary artery followed from ligating the
proximal left anterior descending coronary. We note that
our model focuses primarily on the venous intimal hyper-
plasia. Furthermore our model may be useful for studying
the pathology of vein graft disease and test therapeutic
approaches. Thrombosis, inflammatory cell mirgration
and neointimal hyperplasia in arterialized veins can be
studied in this presented model.
A limitation to this study is the small number of experi-
mental animals. Further studies are therefore necessary.
Furthermore, this model exacts substantial financial cost
and other requirements involved in large animal research.
Small animal models are cheaper, and proper animal care
is easier available compared with large animals.
Conclusion
In conclusion, this new model is suitable for investiga-
tions in accelerated intimal hyperplasia in aortocoronary
internal mammary vein grafts at minipigs. It may support
the investigation of pathophysiology of aortocoronary
vein graft intimal hyperplasia as well as therapeutic
approaches.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AP had helped with surgical techniques, performed data,
analysis, statistics, graphics, and wrorte the paper. JH
Table 1: Dimensional analysis. Luminal area and thickness of the
native mammaria vein and grafted mammaria vein. Thickness
was measured in four regions of interest with respect to the
cardinal of the compass and as mean of the four regions of
interest.
Mammaria vein NativeGraft
p value
Luminal area (mm2)
Intima thickness
North (μm)
East (μm)
South (μm)
West (μm)
Mean (μm)
1.50–12.07(4.25)0.03–26.08(0.96)
n.s.
84–221(120)
74–232(146)
74–253(130)
61–246(142)
61–253(134)
424–2469(1882)
839–2377(1946)
707–2372(1632)
654–2021(1375)
424–2469(1733)
0.001
0.001
0.001
0.001
0.001

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helped with data interpretation and helped to draft the
manuskript. HD, JS and TS and did the surgery on the ani-
mals. VD performed the histological investigations. FS co-
wrote the manuscript and added important comments to
the paper. All authors read and approved the final
manucript.
Acknowledgements
The project was supported by a grant from the Department of Thoracic
Cardiovascular Surgery, University of Göttingen, Germany.
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