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Cardiovascular Research 2003 59(1):222-233; doi:10.1016/S0008-6363(03)00336-5
© 2003 by European Society of Cardiology
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Copyright © 2003, European Society of Cardiology

Effects of intravascular cryotherapy on vessel wall repair in a balloon-injured rabbit iliac artery model

Asim N Cheemaa, Nafiseh Nilia, Christopher W Lia, Heather A Whittinghama, Jacek Lindea, Robert J van Suylenb, Mohammad R Eskandariana, Amy P Wonga, Beiping Qianga, Jean-François Tanguayc, Mimi Laned and Bradley H Straussa,*

aThe 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 M5B 1W8
bDepartment of Pathology, Maastricht University, Maastricht, The Netherlands
cMontreal Heart Institute, Montreal, Canada
dCryoCath® Technologies Inc., Kirkland, Canada

straussb{at}smh.toronto.on.ca

* Corresponding author. Tel.: +1-416-864-5913; fax: +1-416-864-5978.

Received 5 July 2002; accepted 24 February 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
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 –26°C (range, –20 to –45°C) and resulted in significantly larger lumen cross-sectional area (CSA) immediately after application (5.74±1.18 vs. 4.14±0.75 mm2, P = 0.008) 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 (P = 0.01) as well as media/adventitia (P = 0.005) 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.

KEYWORDS Angioplasty; Extracellular matrix; Fibrosis; Restenosis; Calcification


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Vascular response to injury is a critical factor in the 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 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 –30°C 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
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
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 3.0x40 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 3.0x20 mm specially designed catheter for delivery of cryotherapy (CryoCath® Technologies Inc., Kirkland, 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 µg) 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 –30 to –60°C. 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 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 –60°C 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 –30°C (range –20 to –45°C) was achieved in 80% of applications when cryotherapy was applied for 120 s or longer while shorter applications achieved an average temperature of –20°C (range +3 to –41°C).

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).


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  		Table 1 Lumen cross sectional area by IVUS immediately and 10 weeks after cryotherapy

 
2.5 Morphometric measurements and histological analysis
The iliac vessels were embedded in paraffin after fixation and cut into 4 µm 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 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 4x 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
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 40x magnification in five to six representative sections of each artery. The section with the maximum number of neutrophils or macrophages was used for analysis.

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 40x 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 40x 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, ZnCl2 20 mM, NaN3 1.5 mM and SDS 1% w/v). Extracts containing 50 µg 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).

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 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 mean±standard 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
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
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.


Figure 1
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  		Fig. 1 Scheme showing experimental design.

 
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 (583±763 vs. 1994±684 cells/cross-section, respectively, P = 0.003) (Fig. 2A, B). There was also a marked increase in macrophage infiltration in the media of cryotherapy treated arteries (16.2±14.9% vs. 0.5±1.0% in controls, P = 0.02) (Fig. 2C). A prominent neutrophil infiltration (>10/section) was found in two cryotherapy treated arteries but not in any control arteries.


Figure 2
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  		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 40x magnification, hematoxylin and eosin stained sections. (C) Macrophage immunostaining (brown staining nuclei) in cryotherapy treated artery at 72 h. Original 40x magnification. L, lumen; M, media; Ad, adventitia.

 
3.1.2 Cell proliferation
The percentage of proliferating cells was similar in both groups (7.6±5.4% in cryotherapy treated vs. 4.5±4.9% in control, P = ns).

3.1.3 Apoptosis assays
There were no significant differences between control arteries and cryotherapy treated arteries in either TUNEL labeling (0.42±0.36 vs. 4.32±7.83 cells/arterial cross-section, respectively, P = ns). Active caspase-3 levels, indicating apoptosis, were similar in both groups (Fig. 3).


Figure 3
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  		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.

 
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 360° 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.


Figure 4
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  		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.

 
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.

3.2.2 Histomorphometric analysis
3.2.2.1 Reference segment
The lumen and vessel CSA of the proximal reference segment were not significantly different between the treatment groups (Table 2).


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  		Table 2 Morphometric measurements at 10 weeks

 
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 (34.5±8.9 vs. 19.7±5.4 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).


Figure 5
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  		Fig. 5 Increased adventitial (Ad) thickness in cryotherapy treated artery (A) compared to control artery (B) at 10 weeks. Original 4x magnification, I, intima; M, media; L, lumen.

 

Figure 6
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  		Fig. 6 Morphological changes seen in the cryotherapy treated arteries at 10 weeks. (A) Low power (4x) hematoxylin and esoin stained section shows thickening of the arterial wall with a focal region containing bone formation, cartilage and calcification. (B) High power (20x) 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 4x Magnification (D) Original 20x magnification.

 
3.2.4 Cell proliferation
At 10 weeks, cell proliferation was only evident in cryotherapy treated arteries although it was at relatively low levels (2.3±2.2% vs. 0% in controls, P<0.05).

3.2.5 Biochemical analysis
3.2.5.1 Collagen synthesis and content
At 10 weeks, collagen synthesis and content were higher in all treatment 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 collagen synthesis and content compared to control vessels (P≤0.008).


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  		Table 3 Collagen content and synthesis in intima and media/adventitia layers

 
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).


Figure 7
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  		Fig. 7 (A) Correlation between intimal collagen content and late lumen loss. (B) Correlation between media/adventitia collagen content and late lumen loss.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
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 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 {approx}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 (2.15±1.28 vs. 0.56±1.27 mm2, P = 0.001). 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 –60 to –80°C, 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 (–140 to –160°C) 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 by cryotherapy. However, inward remodeling was also an important mechanism since intimal area only accounted for {approx}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 –26°C (range, –20 to –45°C) 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.

Time for primary review 28 days.


   Acknowledgements
 
This study was supported by CryoCath Technologies Inc and is dedicated to the memory of Robyn Strauss Albert.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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T. M. Doherty, L. A. Fitzpatrick, D. Inoue, J.-H. Qiao, M. C. Fishbein, R. C. Detrano, P. K. Shah, and T. B. Rajavashisth
Molecular, Endocrine, and Genetic Mechanisms of Arterial Calcification
Endocr. Rev., August 1, 2004; 25(4): 629 - 672.
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