Cardiovascular Research 59 (2003) 222–233
www.elsevier.com / locate / cardiores
Effects of intravascular cryotherapy on vessel wall repair in a ballooninjured rabbit iliac artery model
Asim N. Cheema a , Nafiseh Nili a , Christopher W. Li a , Heather A. Whittingham a ,
Jacek Linde a , Robert J. van Suylen b , Mohammad R. Eskandarian a , Amy P. Wong a ,
Beiping Qiang a , Jean-François Tanguay c , Mimi Lane d , Bradley H. Strauss a , *
a
The Roy and Ann Foss Interventional Cardiology Research Program, Terrence Donnelly Heart Center, St Michael’ s Hospital, University of Toronto,
30 Bond Street, Toronto, Ontario, Canada M5 B 1 W8
b
Department of Pathology, Maastricht University, Maastricht, The Netherlands
c
Montreal Heart Institute, Montreal, Canada
d
CryoCath  Technologies Inc., Kirkland, Canada
Received 5 July 2002; accepted 24 February 2003
Abstract
Objective: Although the application of cold energy, cryotherapy, has been shown to cause selective damage to cellular components
with preservation of matrix structure resulting in less fibrosis in a variety of tissues, the effects of intravascular cryotherapy on vessel wall
repair after balloon angioplasty are unknown. We sought to characterize the effects of cryotherapy application on vessel wall repair after
balloon angioplasty and study the relationship between collagen accumulation in the vessel wall and late lumen loss as assessed by serial
intravascular ultrasound. Methods: The immediate, early (72 h) and late (10 weeks) effects of three intravascular cryotherapy application
time periods (60, 120 and 240 s) after iliac artery balloon angioplasty (‘cryotherapy’) were compared with balloon angioplasty alone
(‘control’) in 59 rabbits. Arterial lumen area was measured by intravascular ultrasound immediately after the procedure, at 72 h and at 10
weeks. Collagen content was calculated separately for intima and media / adventitia layers and correlated with late lumen loss. Results:
Cryotherapy produced average vessel wall temperature of 226 8C (range, 220 to 245 8C) and resulted in significantly larger lumen
cross-sectional area (CSA) immediately after application (5.7461.18 vs. 4.1460.75 mm 2 , P50.008) but was not different than control
arteries at 10 weeks. At 72 h, there was extensive cell loss in the medial and adventitial layers accompanied by increased macrophage
infiltration in cryotherapy treated arteries compared to control. At 10 weeks, intimal hyperplasia was increased 2-fold in cryotherapy
treated arteries. Collagen content was increased 2-fold in the medial / adventitial layers, and nearly 3-fold in the intima of cryotherapy
treated arteries. Collagen content in arterial intima (P50.01) as well as media / adventitia (P50.005) positively correlated with late lumen
loss. Foci of chondro- and osseous metaplasia and calcification were evident at the medial–adventitial junction in cryotherapy treated
arteries at 10 weeks. Conclusion: Intravascular cryotherapy induced early arterial wall cell loss and late intimal hyperplasia, vascular
fibrosis and chondro- and osseous metaplastic changes with no late beneficial effects on lumen area compared to balloon angioplasty
alone. Collagen accumulation in all three layers of the vessel wall contributes to the development of late inward remodeling after balloon
angioplasty.
 2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved.
Keywords: Angioplasty; Extracellular matrix; Fibrosis; Restenosis; Calcification
1. Introduction
Vascular response to injury is a critical factor in the
*Corresponding author. Tel.: 11-416-864-5913; fax: 11-416-8645978.
E-mail address: [email protected] (B.H. Strauss).
development of several pathological conditions such as
restenosis, atherosclerosis and transplant vasculopathy. In
view of the distinct tissue responses produced by various
physical stimuli, different energy sources have been investigated as an adjunct to balloon angioplasty in an
Time for primary review 28 days.
0008-6363 / 03 / $ – see front matter  2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved.
doi:10.1016 / S0008-6363(03)00336-5
A.N. Cheema et al. / Cardiovascular Research 59 (2003) 222–233
attempt to favorably modulate the reparative process.
Although, the vascular effects of radiation [1,2], laser
[3,4], ultrasound [5,6] and heat [7,8] have been well
characterized, the vascular effects of intravascular application of cold energy are not known. Despite a similar extent
of tissue damage and inflammatory response, freeze-injured nonvascular tissues demonstrate more favorable
healing with preservation of tissue matrix and less wound
contraction compared to burn injury [9]. In liver and skin,
cryotherapy positively influences tissue repair with less
fibrosis [10–12]. This favorable healing response has been
attributed to selective injury to cellular components and
preservation of collagen fiber network and matrix structure
in the cryotherapy treated tissue [11,12].
Arterial repair after balloon angioplasty is also characterized by a fibrotic response in the vessel wall [13,14].
Recently, a 5F flexible catheter has been developed that
can deliver temperatures below 230 8C to the vessel wall
with intraluminal placement. We hypothesized that
cryotherapy application after balloon angioplasty would
inhibit this fibrotic response and favorably alter arterial
healing, as seen in other tissues, and prevent late loss of
lumen area. In the present study, we sought to assess the
early (72 h) and late (10 weeks) effects of intravascular
cryotherapy application on arterial repair in a rabbit
balloon angioplasty model. In addition, we intended to
delineate mechanisms of lumen area loss after balloon
injury by prospectively determining the relationship between collagen accumulation in the various layers of the
vessel wall and late loss of lumen area assessed by serial
intravascular ultrasound studies.
2. Methods
2.1. The animal model and study protocol
The animal experiments were performed in accordance
with the Guide for the Care and Use of Laboratory
Animals published by the US National Institute of Health
and approved by the St. Michael’s Hospital Animal Care
Committee. We used normolipemic male New Zealand
white rabbits weighing 3.5–4.00 kg. Two time periods
were studied, at 72 h after injury (early response) and at 10
weeks after injury (late response). In the former, a single
injury model (SIM) was used, while the 10-week study
was performed using the double injury model (DIM) as
previously described [13,14]. In both injury models, balloon injury was performed in the iliac artery, beginning 3
mm beyond the aortic bifurcation by inflating a 3.0340
mm length angioplasty balloon four times, with four 1-min
inflations (6, 8, 4 and 10 atmospheres). This injury was
repeated 3 weeks later in the DIM. Immediately after the
first injury in the SIM and after the second injury in the
DIM, a 3.0320 mm specially designed catheter for delivery of cryotherapy (CryoCath  Technologies Inc., Kirk-
223
land, Canada) was advanced and placed serially in both
iliac arteries within the balloon injured segment.
Cryotherapy was applied to the vessel wall according to a
predetermined protocol (see below). Intra-arterial nitroglycerin (150 mg) was administered followed by intravascular ultrasound (IVUS) study of the treated and
proximal reference segments using a 2.9 F, 30 MHz
intravascular ultrasound catheter (Ultracross, Boston Scientific). At 72 h after injury in the SIM and at 10 weeks
after the second injury in the DIM, rabbits were again
anesthetized and access was obtained in bilateral femoral
arteries. An iliac angiogram was performed after administration of intra-arterial nitroglycerin to relieve any vasospasm. Intravascular ultrasound catheter was introduced
through one femoral artery and advanced to the aortic
bifurcation. The catheter was then slowly pulled back and
ultrasound images continuously recorded from origin of
iliac artery to proximal femoral arteries. The intravascular
ultrasound was then completed on the contralateral side in
the similar fashion. After acquisition of intravascular
ultrasound images, the femoral arteries were ligated and
the abdomen was surgically opened. For biochemical
studies, iliac artery tissue was removed under general
anaesthetic, followed by a fatal intracardiac injection of
thiopental. For histomorphometric analysis of the 10-week
arteries, perfusion fixation was done with 10% neutral
buffered formalin for 30 min at a perfusion pressure of 80
mmHg through a 5F sheath placed in the descending aorta.
2.2. Cryotherapy application
A 5F over-the-wire flexible catheter (CryoCath) was
used for intravascular cryotherapy application. The catheter has two closed lumens in addition to a central lumen
for passage of guide wire and temperature is recorded from
the distal tip with integrated thermocouples. The console
delivers the cooling fluid, AZ20 (Genetron  ), to the distal
tip where a liquid to gas phase change results in distal
catheter temperatures of 230 to 260 8C. This gas is
conducted away from the tip through the vacuum return
lumen and is collected in the console. To determine the
effects of different doses of cryotherapy application on the
arterial wall following balloon injury, iliac arteries were
assigned to one of the four treatment groups. The
cryotherapy delivery catheter was placed in iliac vessels in
the ‘control’ group but no cryotherapy was applied (balloon angioplasty alone). ‘Low dose’ treatment group had
cryotherapy application for 60 s and ‘intermediate dose’
group had cryotherapy application for 120 s. Cryotherapy
was applied for 240 s in the ‘high dose’ group. In the DIM,
all three doses were compared to controls. In the SIM, only
the high dose group was compared to controls.
2.3. Temperature determination study
In order to determine temperatures achieved at the inner
A.N. Cheema et al. / Cardiovascular Research 59 (2003) 222–233
224
vessel wall during intraluminal cryotherapy, two rings of
four thermocouples were placed on metallic stents, which
were then deployed in 10 rabbit iliac arteries. Intravascular
cryotherapy was delivered for 60, 120 and 180 s as
described earlier and temperatures were recorded from the
stents via the thermocouples as well as from the delivery
catheter tip during cryotherapy application. Although a
temperature of 260 8C recorded from catheter tip was
achieved in all applications, the temperature recordings
from stent thermocouples varied with the total duration of
application. Vessel wall temperature below 230 8C (range
220 to 245 8C) was achieved in 80% of applications
when cryotherapy was applied for 120 s or longer while
shorter applications achieved an average temperature of
220 8C (range 13 to 241 8C).
measurements. Morphometric measurements were performed on six to eight representative sections of proximal
reference and the treated segments. For determination of
vessel dimensions, the sections were viewed at 43 with
BX-50 Olympus microscope and images transferred to a
compact disc with CoolSNAP  camera and software for
offline analysis. All measurements were completed using
the Scion image  imaging software. An observer blinded
to the treatment group assignment measured the lumen,
intimal and vessel (external elastic lamina) CSA for the
proximal reference and the treated segments. The intimal
area was calculated by deducting the luminal area from the
area defined by the internal elastic lamina.
2.6. Collagen and elastin determination
2.4. Intravascular ultrasound measurements
All ultrasound images recorded on VHS videotape were
analyzed offline. A blinded observer calculated vessel
dimensions with a digital video analyzer during manual
pullback and at regular intervals. Lumen cross sectional
area (CSA) was measured for the proximal reference
segment (the 3 mm segment distal to the aortic bifurcation
that was not injured) as well as at three sites (proximal,
middle and distal) within the cryotherapy treated segment
of each vessel. Lumen CSA of the treated segment was
determined by calculating the mean of the measurements
of the proximal, middle and distal CSA. The intravascular
ultrasound (IVUS) measurements were performed immediately after cryotherapy application and prior to sacrifice
(Table 1).
2.5. Morphometric measurements and histological
analysis
The iliac vessels were embedded in paraffin after
fixation and cut into 4 mm thick sections. The hematoxylin
and eosin stained sections were used for histological
examination under light microscopy while Movat pentachrome stained sections were used for morphometeric
In arteries removed 10 weeks after angioplasty, the
medial / adventitial layers were manually separated from
the intima in the 15 mm mid segment of selected
cryotherapy and control iliac arteries and the layers were
separately analyzed for collagen and elastin synthesis and
content as described previously [13]. The adequacy of the
separation of the vessel wall layers by this method has
been previously validated [14].
2.7. Assessment of tissue inflammation
Arterial cross sections from both 72 h and 10 weeks
animals were stained for the presence of neutrophils and
macrophages. Mouse monoclonal antibodies against rabbit
neutrophils (1 / 50 dilution, Serotec Inc., NC, USA) and
rabbit macrophages, RAM11 (1 / 50 dilution, DAKO, CA,
USA) were used on paraffin embedded sections and
examined under light microscopy. The total number and
percentage of neutrophils and macrophages in arterial
cross-sections were counted at 403 magnification in five
to six representative sections of each artery. The section
with the maximum number of neutrophils or macrophages
was used for analysis.
Table 1
Lumen cross sectional area by IVUS immediately and 10 weeks after cryotherapy
Control
Low dose
Intermediate dose
High dose
All Cryo doses
Reference segment (mm )
Immediate
10 weeks
5.5961.48
5.2461.33
5.6761.52
5.1961.47
5.7861.50
5.4361.21
5.7661.22
5.0561.37
5.7361.41
5.2261.35
Treated segment (mm 2 )
Immediate
10 weeks
4.1460.75
3.5861.00
5.5961.39 a
3.5160.45
5.7860.92 a
3.5760.81
5.8461.23 a
3.6760.78
5.7461.18 a
3.5860.68
Late loss (mm 2 )
0.5661.27
2.0861.43 b
2.2161.12 b
2.1761.29 b
2.1561.28 b
2
Data are expressed as mean6S.D. Cryo, cryotherapy.
a
P#0.01 compared to control.
b
P#0.003 compared to control.
A.N. Cheema et al. / Cardiovascular Research 59 (2003) 222–233
2.8. Cell proliferation
Proliferating cells both in control and cryotherapy
treated arteries were identified by immunohistochemistry
using a mouse anti rabbit monoclonal antibody directed
against Mib-1 [15] (1 / 500 dilution, DAKO) at 72 h and at
10 weeks after angioplasty. Mib-1 positive cells were
counted using an image analysis system (Scion Image,
Scion Corp) under 403 magnification. Proliferation rates
were expressed as the percentage (%) of Mib-1 positive
cells in the arterial cross-section.
2.9. Apoptosis assays
2.9.1. TUNEL assay
Apoptotic cells were identified in cryotherapy treated
and control arteries at 72 h after angioplasty using TUNEL
assay (terminal deoxy nucleotidyl transferase mediated
Nick End Labeling). The TUNEL assay was performed
according to manufacturer’s instructions (ApopTag 
Fluorescein In Situ Apoptosis Detection Kit-INTERGEN,
NY, USA). Sections pretreated with DNase 1 served as a
positive control. Negative controls consisted of staining for
DNA strand breaks without TdT (linker enzyme) but
including proteinase K digestion to control for non-specific
binding of enzyme conjugate. Apoptotic cells were identified at 403 magnification and expressed as a % of the
total number of nuclei in the arterial cross-section.
2.9.2. Caspase-3 Western blotting
Frozen sections of arteries removed at 72 h after
angioplasty were pulverized in liquid nitrogen and extracted in ice-cold extraction buffer (cocodylic acid 10
mM, NaCl 150 mM, ZnCl 2 20 mM, NaN 3 1.5 mM and
SDS 1% w / v). Extracts containing 50 mg protein were
analyzed by 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and electroblotted onto a nitrocellulose membrane. The membrane
was immunoblotted with rabbit polyclonal anti caspase-3
(CPP32) Ab-4 (NeoMarker, Fremont, CA, USA). Anti
rabbit IgG-HRP (Santa Cruz Biotechnology) was used for
detection of primary antibody and revealed using chemiluminescence detection system (Sigma, St. Louis, MO,
USA) followed by autoradiography using BioMax film
(Kodak, Rochester, NY, USA).
225
staining was confirmed by substituting the primary antibody with normal goat serum at the same dilution.
2.11. Statistical analysis
All measurements were expressed as mean6standard
deviation (S.D.). The collagen and elastin synthesis and
content were compared between the four treatment groups
by ANOVA. Bonferroni’s correction was applied for
multiple comparisons. Student’s t-test was used when data
was compared for only two treatment groups (all
cryotherapy treated arteries combined and control arteries).
The categorical data were compared with Chi square or
Fisher Exact as appropriate. Pearson’s correlation coefficient was used to determine the relationship between
intimal hyperplasia and late lumen loss as well as collagen
content in the intima and media / adventitia and late lumen
loss. Statistical significance was defined as P#0.05.
3. Results
A total of 59 animals were included in the study. The
details of vessels available for IVUS analysis, histology,
biochemical assays and morphometry are shown in Fig. 1.
Four rabbits died before completion of end study. These
animals died at 20 min, 60 min and at 3 days and 15 days
after cryotherapy application.
3.1. Effects of cryotherapy at 72 h
3.1.1. Histopathological analysis
The main feature was presence of extensive cell loss
throughout the media and the adventitia in seven of the
nine cryotherapy treated arteries while only one of the six
control arteries showed evidence of moderate cell loss
which was restricted to a third of the arterial circumference. The mean cell number in the media was significantly
lower in cryotherapy treated arteries compared to control
arteries (5836763 vs. 19946684 cells / cross-section, respectively, P50.003) (Fig. 2A, B). There was also a
marked increase in macrophage infiltration in the media of
cryotherapy treated arteries (16.2614.9% vs. 0.561.0% in
controls, P50.02) (Fig. 2C). A prominent neutrophil
infiltration (.10 / section) was found in two cryotherapy
treated arteries but not in any control arteries.
2.10. Staining for bone morphogenic protein ( BMP)
Arteries removed at 10 weeks after angioplasty were
examined for immunohistochemical localization of BMP2.
Arterial sections were incubated with a goat polyclonocal
antibody against BMP2 (RDI-BMP2Nabg, Research Diagnostics Inc., NJ, USA) at a dilution of 1:500. A secondary
anti-goat antibody was used. The specificity for BMP2
3.1.2. Cell proliferation
The percentage of proliferating cells was similar in both
groups (7.665.4% in cryotherapy treated vs. 4.564.9% in
control, P5ns).
3.1.3. Apoptosis assays
There were no significant differences between control
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A.N. Cheema et al. / Cardiovascular Research 59 (2003) 222–233
Fig. 1. Scheme showing experimental design.
Fig. 2. Morphological changes seen in a cryotherapy treated (A) and a balloon only treated (B) artery at 72 h. There is marked cell loss in the media and
the adventitia of the cryotherapy treated artery. Original 403 magnification, hematoxylin and eosin stained sections. (C) Macrophage immunostaining
(brown staining nuclei) in cryotherapy treated artery at 72 h. Original 403 magnification. L, lumen; M, media; Ad, adventitia.
A.N. Cheema et al. / Cardiovascular Research 59 (2003) 222–233
arteries and cryotherapy treated arteries in either TUNEL
labeling (0.4260.36 vs. 4.3267.83 cells / arterial crosssection, respectively, P5ns). Active caspase-3 levels,
indicating apoptosis, were similar in both groups (Fig. 3).
3.2. Effects at 10 weeks
3.2.1. Intravascular ultasound analysis
3.2.1.1. Acute
change
in
tissue
acoustic
characteristics An echolucence was evident at the medial / adventitial border in cryotherapy treated segments
immediately after cryotherapy application that was not due
to presence of dissections (Fig. 4A, B). This echolucent
area covered only a few degrees in some arteries but
extended to 3608 of circumference in other cryotherapy
treated arteries. These echolucent areas were transiently
present after the cryotherapy application and diminished
over a 20-min time period. This change in tissue acoustic
characteristics was more common in the high dose
cryotherapy group and limited to the treated segment only.
These effects were not observed in the control arteries.
There was no significant change in the lumen CSA of
the proximal reference segment between the four treatment
groups immediately after cryotherapy or at 10 weeks. The
lumen CSA of the treated segment was significantly larger
immediately after cryotherapy application in all
cryotherapy treated groups compared to control vessels.
However, at 10 weeks, there was no significant difference
in lumen CSA between control and cryotherapy treated
vessels. The late lumen loss was therefore significantly
higher in cryotherapy treated vessels compared to controls.
227
of the proximal reference segment were not significantly
different between the treatment groups (Table 2).
3.2.2.2. Treated segment At 10 weeks, the lumen and
vessel CSA was not significantly different between
cryotherapy treated vessels compared to controls. The
mean intimal area of treated segments in each of three
cryotherapy groups was significantly increased compared
to control (P#0.003).
3.2.3. Histopathological analysis
Gross examination at harvesting of iliac vessels showed
significant fibrosis and adhesions around the cryotherapy
treated vessels that were not present in control vessels.
This fibrotic response was limited to the treated segment
and did not extend proximally or distally along the vessel
length. The wet weight of the treated segments was
significantly higher in cryotherapy treated vessels compared to controls (34.568.9 vs. 19.765.4 mg / arterial
segment, P,0.001).
The cryotherapy treated vessels showed a significantly
increase in both intimal and adventitial layer thickness
compared to controls (Fig. 5). The vessel wall of the
cryotherapy treated (medium and high dose group) arteries
also showed presence of areas containing cartilage, bone
tissue and calcification in the media and at the medial–
adventitial junction that were not seen in any control
arteries (Fig. 6A, B). Immunohistochemistry showed presence of bone morphogenic protein (BMP2) in areas
containing calcification (Fig. 6C, D).
3.2.4. Cell proliferation
At 10 weeks, cell proliferation was only evident in
cryotherapy treated arteries although it was at relatively
low levels (2.362.2% vs. 0% in controls, P,0.05).
3.2.2. Histomorphometric analysis
3.2.5. Biochemical analysis
3.2.2.1. Reference segment
3.2.5.1. Collagen synthesis and content At 10 weeks,
collagen synthesis and content were higher in all treatment
The lumen and vessel CSA
Fig. 3. Western blot analysis of caspase-3 in arteries at 72 h. There are similar levels of active caspase-3, indicating cell apoptosis, in both cryotherapy
treated and control (balloon only treated) arteries.
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A.N. Cheema et al. / Cardiovascular Research 59 (2003) 222–233
Fig. 4. (A) Intravascular ultrasound arterial images immediately after balloon treatment (left hand panel) and cryotherapy treatment (right hand panel).
Cryotherapy treated artery shows marked increased in lumen (L) compared to the balloon treated artery. An echolucent area is also evident at the
media / adventitia border (indicated by arrow) immediately after cryotherapy application. IVUS catheter is labeled as ‘C’. (B) The histologic section of the
cryotherapy treated segment from (A), showing absence of dissection or hematoma in the vessel wall.
groups compared to controls. The collagen synthesis and
content in the intima of high dose cryotherapy treated
vessels showed a 6- and 4-fold increase, respectively,
compared to control (P#0.01) (Table 3). Similarly, the
medial / adventitial layer of the cryotherapy treated vessels
also demonstrated a greater than 2-fold increase in col-
A.N. Cheema et al. / Cardiovascular Research 59 (2003) 222–233
229
Table 2
Morphometric measurements at 10 weeks
Control
Low dose
Intermediate dose
High dose
All Cryo doses
Reference segment (mm 2 )
Lumen CSA
Vessel (EEL) CSA
1.6660.94
2.5361.06
1.4060.74
2.2360.74
1.7560.98
2.7661.22
1.6660.59
2.6660.73
1.6660.59
2.5560.89
Treated segment (mm 2 )
Lumen CSA
Vessel (EEL) CSA
Intimal area
Medial area
Adventitia area
1.6760.43
2.4160.59
0.2660.15
0.3960.06
0.3560.08
1.7960.57
2.7560.61
0.5260.17 a
0.4160.10
0.4960.12
1.8260.42
2.8860.39
0.5960.13 a
0.4660.09
0.4760.09
1.9660.39
3.0060.48
0.5360.25 a
0.5060.11
0.5760.24 b
1.8560.46
2.8160.49
0.5460.18 a
0.4660.10
0.5160.16 b
Data are expressed as mean6S.D. Cryo, cryotherapy; EEL, external elastic lamina; CSA, cross sectional area.
a
P#0.003 compared to control.
b
P#0.007 compared to control.
lagen synthesis and content compared to control vessels
(P#0.008).
3.2.5.2. Elastin synthesis and content Elastin synthesis
and content were not significantly different between the
cryotherapy treated and control arteries (data not shown).
3.2.6. Relationship between collagen content and lumen
loss by IVUS
The collagen content of the vessel wall showed a
significant correlation with late lumen loss in the treated
segments. This positive relationship between increased
collagen content and late lumen loss was present for both
the intimal and medial / adventitial layers (Fig. 7A, B).
4. Discussion
In this study we report for the first time the early and
late arterial effects of intravascular cryotherapy after
balloon angioplasty. In contrast to previous reports of
decreased fibrosis in skin and liver lesions treated with
cryotherapy [10–12], the main findings of this study are
that intravascular cryotherapy after balloon angioplasty
resulted in an early response characterized by marked cell
loss in the medial and adventitial layers. This appears to be
due to predominantly cell necrosis as evidenced by the
prominent macrophage infiltration and enhanced inflammatory response. There was minimal evidence of active
apoptosis in either group based on the caspase-3 western
immunoblotting and TUNEL labeling studies. This early
response was followed by a late, aggressive fibrotic
response with a marked increased in collagen accumulation
in all layers of the vessel. As a consequence, there was no
difference in lumen area at 10 weeks compared to control
balloon treated arteries.
This neutral late effect on lumen area occurred despite a
significantly larger lumen area immediately after
cyotherapy in all three cryotherapy treated groups compared to control balloon treated arteries. The extent of this
Fig. 5. Increased adventitial (Ad) thickness in cryotherapy treated artery (A) compared to control artery (B) at 10 weeks. Original 43 magnification, I,
intima; M, media; L, lumen.
A.N. Cheema et al. / Cardiovascular Research 59 (2003) 222–233
230
Fig. 6. Morphological changes seen in the cryotherapy treated arteries at 10 weeks. (A) Low power (43) hematoxylin and esoin stained section shows
thickening of the arterial wall with a focal region containing bone formation, cartilage and calcification. (B) High power (203) enlargement of Fig. 3A. L,
arterial lumen; B, bone tissue; C, cartilage tissue; Ca, calcification. (C) Presence of bone morphogenic protein (BMP2) in areas of calcification of
cryotherapy treated arteries is shown as brown staining areas in the arterial wall. Original 43 Magnification (D) Original 203 magnification.
Table 3
Collagen content and synthesis in intima and media / adventitia layers
Control
Low dose
Intermediate
High dose
All Cryo doses
139679
1866163
111678
2356118 b
2776135 a
2726102 c
176697 a
2316127 b
93648
266691 d
95649
265665 d
133650 c
278680 d
107649 c
270679 d
Dose
Collagen synthesis (cpm
Intima
Media / adventitia
14
C-hydroxyproline / segment)
36630
50668
Collagen content (mg hydroxyproline / segment)
Intima
39621
Media / adventitia
13669
Data are expressed as mean6S.D. Cryo, cryotherapy. P5ns where not mentioned.
a
P,0.001 compared to control and P#0.03 compared to low and intermediate dose.
b
P#0.04 compared to control.
c
P#0.008 compared to control.
d
P#0.01 compared to control.
A.N. Cheema et al. / Cardiovascular Research 59 (2003) 222–233
231
Fig. 7. (A) Correlation between intimal collagen content and late lumen loss. (B) Correlation between media / adventitia collagen content and late lumen
loss.
lumen enlargement was quite dramatic and comparable to
the acute results seen post stent implantation in this animal
model [14]. The exact mechanism of this acute gain is
unclear but it is possible that cryotherapy prevents acute
recoil by disturbance of autonomic regulation due to
damage to vascular nerves, direct or indirect local release
of vasodilator substances or due to the marked cell
necrosis within the medial and adventitial layers of vessel
wall. Serial IVUS measurements indicated that ¯50% of
the acute gain was still persistent at 72 h (data not shown).
Holman et al. [16] noted hyperemic response and significant increase in blood flow measured by electromagnetic
flow probe after external cryotherapy application on proximal LAD in dogs although lumen dimensions were not
measured in this study.
Despite this significant increase in early acute gain after
cryotherapy application, the lumen area at 10 weeks was
similar in all treatment groups, due to a 4-fold increase in
late lumen loss in cryotherapy treated arteries compared to
control balloon treated arteries (2.1561.28 vs. 0.5661.27
mm 2 , P50.001). Intravascular cryotherapy application
after balloon angioplasty resulted in significantly increased
intimal hyperplasia in the present study. Vascular effect of
cryotherapy have been described in several studies of
cryotherapy application to the myocardium for the treatment of arrhythmias [16–18]. In addition, a few studies
have also evaluated the vascular responses after local
external cryotherapy application using liquid nitrogen [19].
These studies report variable effects on intimal hyperplasia
and are difficult to compare due to the wide range of
temperatures and application times used with different
cooling agents in diverse animal models. With temperatures of 260 to 280 8C, Bertelli et al. [19] reported
absence of a neointimal response in rat femoral arteries
using liquid nitrogen spray, while Iida et al. [18] reported
moderate intimal thickening at 1 month that regressed at 6
months in dog coronary arteries with similar temperatures.
In contrast, much colder temperatures (2140 to 2160 8C)
also using liquid nitrogen have been shown to produce
moderate intimal hyperplasia in rat femoral arteries [20] as
well as coronary arteries of larger animals [21,22]. There is
only one report on arterial effects after human His bundle
cryoablation which described fibrinoid necrosis and
atheroma-like intimal thickening in the AV nodal coronary
artery from autopsy specimens at 1 and 6 weeks [23].
A novel observation in the present study was the
presence of cartilage, bone formation, and calcification in
the arterial media of the cryotherapy treated segments.
These changes were seen in 60–70% of the intermediate
and high dose cryotherapy group. Although myocardial
application of cryotherapy in animal models has been
shown to produce chondroid metaplasia and focal calcification in the myocardium [21,24,25], no such abnormalities
were reported in the vessel wall. The presence of chondroblast, osteoblast and calcium deposition seen in the
vessel wall after cryotherapy are features of endochondral
osteogenesis and are seen in growth plate development, in
fracture healing [26] or in fibrodysplasia ossificans progressiva [27]. The arterial segments displaying bone
formation also stained positive for the extracellular bone
matrix protein, BMP2, demonstrating active cartilage and
bone formation in contrast to passive calcium deposition or
calcification in hemorrhagic areas [28]. The majority (2 / 3)
of cryotherapy treated arteries demonstrated nearly complete cell loss throughout the media and the adventitia at
72 h. There was also a prominent macrophage infiltration
in cryotherapy treated arteries, which likely explains the
morphologic changes (bone and cartilage formation) seen
in the vessel wall at 10 weeks. The macrophages express
BMP [29] and can also activate vascular pericytes, the
precursor cells known to express osteoblastic markers and
cause bone formation both in vitro and in vivo [30,31].
4.1. Collagen accumulation and late loss of lumen area
As described earlier, intimal hyperplasia was stimulated
232
A.N. Cheema et al. / Cardiovascular Research 59 (2003) 222–233
by cryotherapy. However, inward remodeling was also an
important mechanism since intimal area only accounted for
¯25% of the late loss of lumen area in cryotherapy treated
arteries (and 46% of lumen area loss in control balloon
treated arteries). The wide range of collagen contents and
lumen loss across the four experimental groups presented a
unique opportunity to critically examine the relationship
between collagen accumulation and lumen loss. There
were two important components of the collagen response
to arterial injury. First, we showed a significant correlation
between intimal collagen content and lumen loss, which is
not surprising since we have previously demonstrated that
intimal collagen is an important component of intimal
hyperplasia [13,14]. However, we also demonstrate a
significant positive correlation between collagen content in
the medial / adventitial layers and late lumen loss, providing the strongest evidence to date of the role of medial /
adventitial collagen accumulation in late inward remodeling. Two previous studies of collagen accumulation and
arterial remodeling [32,33] have produced inconsistent
results. Lafont et al. [32] also demonstrated a positive
correlation between intimal collagen accumulation in the
vessel wall and inward remodeling, while Coats et al. [33]
reported that increased collagen content was associated
with positive remodeling and thus prevention of restenosis.
The present study has several important methodological
differences compared to these two studies. We included a
larger number of arteries with a wide range of collagen
content values, which were quantitatively determined
separately for intima and media / adventitia layers. Accurate assessment of late lumen loss by IVUS in our study
also avoided the pitfalls of histological measurements,
particularly vessel shrinkage associated with tissue processing.
In conclusion, despite improved acute effects on lumen
enlargement, the application of intravascular cryotherapy
with average temperature of 226 8C (range, 220 to
245 8C) at the vessels wall after balloon angioplasty
results in increased intimal hyperplasia with an enhanced
fibrotic response in all layers of the arterial wall.
Cryotherapy resulted in severe arterial wall cell loss at the
early time period that was followed by late morphological
changes, including cartilage and bone formation as well as
localized calcification were present in the vessel wall of
cryotherapy treated arteries. Collagen accumulation in all
three layers of the vessel wall plays an important role in
the development of late inward remodeling as demonstrated by a positive correlation between intimal as well as
medial / adventitial collagen content and late lumen loss
after balloon angioplasty.
Acknowledgements
This study was supported by CryoCath Technologies Inc
and is dedicated to the memory of Robyn Strauss Albert.
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