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1 Department of Vascular Surgery, 2 Department of Interventional Radiology, Rechts der Isar Medical Center, Technical University of Munich, Germany
Correspondence: Dr Peter Heider, Department of Vascular Surgery, Rechts der Isar Medical Center, Technical University of Munich, Ismaninger Strasse 22, D-81675 Munich, Germany. E-mail: heiderpeter{at}t-online.de
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Abstract |
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) and basic fibroblast growth factor (bFGF)) and the cytokine tumour necrosis factor alpha (TNF
) after both angioplasty and cryoplasty. Recently cryoplasty has been suggested as a new method to oppose neointimal hyperplasia resulting in restenosis formation. While in vitro models have shown that the application of cryothermal energy to the endothelium during angioplasty leads to apoptosis induction and reduced proliferation rates, no human in vivo proof for an inhibition of neointimal hyperplasia exists. For restenosis initiation adhesion molecules, growth factors and cytokines play an important role. One possibility to investigate the endothelial response to angioplasty is the measurement of the soluble forms of adhesion molecules, growth factors and cytokines that are released into the circulation after denuding the vessel wall. In the present study we assessed the distribution pattern of the soluble forms of e-selectin, p-selectin, ICAM, VCAM, MCP-1, TGF, bFGF and TNF after angiography, angioplasty and cryoplasty of the femoropopliteal artery in the early course of 4 weeks in 29 patients with peripheral arterial occlusive disease. During the 4 weeks after intervention levels of e-selectin, ICAM, VCAM and MCP-1 increased after both angioplasty and cryoplasty. The course of the screened biomarkers was similar between angioplasty and cryoplasty. P-selectin and TGF both decreased after cryoplasty, but not significantly. The present results show that the release of adhesion molecules, growth factors and cytokines is similar between balloon angioplasty and cryoplasty. |
Introduction |
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Interventional radiologists and vascular surgeons may now potentially limit restenosis by the use of cryoplasty that combines balloon angioplasty with the delivery of cryothermal energy to the endothelium. Despite first promising results [2, 3], little is known about the cellular and humoral mechanisms that might result in reduced neointimal hyperplasia and lower restenosis rates.
In our study we investigated prospectively the distribution pattern of a variety of soluble cell adhesion molecules (intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), e-selectin, p-selectin, and monocyte chemoatlractant protein 1 (MCP-1)), growth factors (transforming growth factor beta (TGF) and basic fibroblast growth factor (bFGF)) and cytokines (tumour necrosis factor alpha (TNF)) that are considered to play an important role in mediating restenosis development after peripheral angioplasty [4–7]. These factors have a predictive value for restenosis formation and therefore may also be markers in the assessment of cryoplasty [8, 9].
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Methods and materials |
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Patients
29 patients (13 men, 16 women, mean age 70.3±7.1 years) with peripheral arterial occlusive disease (PAOD) and indication for interventional procedure were studied. All patients presented with symptoms of intermittent claudication (Fontaine stage IIb). The severity of PAOD was assessed by measurement of ankle brachial index (ABI) at rest and treadmill test. Morphological graduation of the lesions was performed non-invasively by duplex ultrasound (System Five, Vingmed Sounds A/S, Horten, Norway) and invasively during angiography. The patient characteristics are listed in Table 1
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Lesions
29 patients with lesions in the femoropopliteal artery were enrolled in the study. 21 patients (72.4%) had stenosis, 8 patients (27.6%) had occlusion of the artery. In patients with stenotic lesions the average lumen narrowing was 81.9±13.7%, graded on angiographic profile by the performing radiologist. Detailed descriptions of lesions are shown in Table 2.
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Angiography and angioplasty procedures were carried out according to established and accepted techniques [11]. The decision whether cryoplasty or angioplasty was performed in a patient was made by the performing radiologist based on the morphology and extent of the arterial lesion in the angiographic picture. Since only a restricted selection of cryoplasty catheters concerning size and lumen of the catheter is available, some lesions required angioplasty when cryoplasty could not be performed due to the size of the lesion. Thus, the assignment of the patients to either angioplasty or cryoplasty was not randomized.
Cryoplasty procedures were performed with PolarCath balloon angioplasty catheters (CryoVascular Systems, Los Gatos, CA). During angioplasty liquid nitrous oxide flows through the triple lumen catheter to the balloon and changes to a gaseous state, expanding the balloon and lowering the temperature. The entire process exposes the target lesion to an algorithm of temperature (–10°C), pressure (8 atm) and dwell time (20 s). After completion of the cycle, the gas is evacuated deflating the balloon. The balloon can then be removed or an additional cycle can be repeated.
Altogether 20 balloon angioplasty procedures were performed in 12 patients (four patients received only a single balloon angioplasty, eight patients required a second dilation). In the cryoplasty group (n = 8) two patients were treated with a single cryoplasty, five patients required a second cryoplasty, one patient was treated with five cryoplasties in the same target vessel, so that altogether 17 cryoplasties were performed.
The interventions were all carried out by two experienced interventional radiologists (WW, HB). All interventional procedures were technically successful. A bolus of 3000–5000 IU unfractionated heparin was given intra-arterially as antithrombotic prophylaxis. After PTA acetylsalicylate acid 100 mg day–1 and clopidogrel 75 mg day–1 were given as standard treatment. Further characteristics of the performed procedures are listed in Table 2.
Laboratory tests
Venous blood samples for laboratory testing were drawn in pyrogen-free tubes on the day before the interventional procedure (between 10 am and 12 noon), 15 and 60 min after the last inflation of the angioplasty balloon or the cryoplasty balloon, 24 h after the intervention, as well as 2 and 4 weeks after the intervention. In patients who received only the angiographic procedure, the samples were obtained on the day before the procedure, 15 and 60 min after the last injection of contrast material, 24 h after angiography as well as 2 and 4 weeks after the intervention. Blood samples were immediately centrifuged at 2500 rpm for 5 min and obtained serum was stored at –70°C until laboratory testing. According to the results of Pai et al [12] who investigated the stability of novel plasma markers in different time ranges from specimen collection to processing, the time range from sample collection until freezing was a maximum of 2 h.
Laboratory testing for e-selectin, p-selectin, ICAM, VCAM, MCP-1, bFGF, TGF and TNF was performed using standardized enzyme-linked immunosorbent assay (ELISA) kits (BioSource International, Camarillo, CA). Standardization was done using recombinant factors provided with the kits. An ELISA reader (Dynatech Laboratories, USA) set to 450 nm with wavelength correction set to 620 nm determined the optical density. The minimum detectable levels were 1.3 ng ml–1 for p-selectin, 0.5 ng ml–1 for e-selectin, 0.9 ng ml–1 for VCAM-1, 0.5 ng ml–1 for ICAM-1, 20 pg ml–1 for MCP-1, 15.6 pg ml–1 for TGF 1.7 pg ml–1 for TNF and 7 pg ml–1 for bFGF.
Additionally we investigated whether the freezing of the target lesion causes increased haemolysis in the blood proximal to the cryoplasty balloon. Therefore arterial blood samples were drawn through the sheath system from the arterial segment proximal to the balloon exactly 15 s after the cryoplasty balloon had been inflated by the cooled nitric oxide. Arterial blood samples were screened for lactate dehydrogenase (LDH) and electrolytes (sodium, potassium, calcium) and compared with values obtained before cryoplasty as well as with values 5 min after cryoplasty when the catheter was removed and the blood flow restored.
The performing radiologists were blinded to the laboratory results.
Statistics
Results are given as mean ± standard deviation. Group comparisons of patient characteristics were done with the &Chi-
2 test and Kruskal–Wallis test. Analysis of medication and risk factors was planned as univariate analysis in consideration for the small observation group. Comparisons of soluble factor levels over the time course were done with two-factor analysis of variance (Anova) (one between, one within). Comparisons of the factor levels between the intervention groups (angiography, angioplasty and cryoplasty) were performed with repeated Anova.
A probability level of 0.05 or less (p<0.05) was considered to be statistically significant. All analyses were performed with SPSS 12.0 (SPSS Inc., Chicago, IL).
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Results |
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. Sex distribution was different in patients being treated with cryoplasty (one male, seven female, p = 0.025). Concerning all other characteristics patients were comparable between the intervention groups.
Lesions
Angiographic and procedural characteristics are shown in Table 2. The majority of the treated lesions were femoral lesions Type TASC A and B (p = 0.003). Patients receiving angiography only had significantly longer lesions than patients treated with angioplasty or cryoplasty (p = 0.026). Otherwise the intervention groups did not differ significantly between each other.
Levels of soluble markers
The levels of TNF and bFGF were below the detection limit at all time points in patients undergoing angiography, angioplasty or cryoplasty. Serum concentrations above the detection limit of p-selectin, e-selectin, ICAM, VCAM, MCP-1 and TGF were found in all groups. Significant changes over the observation period were detected in p-selectin (p = 0.001), e-selectin (p = 0.023), ICAM (p = 0.007), MCP-1 (p = 0.001) and TGF (p = 0.007); the shift of VCAM levels reached only borderline significance (p = 0.073) (see Table 3).
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ICAM and VCAM
While serum levels of ICAM and VCAM remained stable in patients undergoing angiography only, ICAM and VCAM levels increased in patients both with angioplasty and cryoplasty to reach higher levels at 4 weeks after intervention (p = 0.007 and p = 0.073). Serum levels of ICAM and VCAM did not differ significantly between cryoplasty and angioplasty over the whole observation period (repeated Anova: p = 0.409 and p = 0.851).
MCP-1
MCP-1 levels increased significantly in the 4 week course after cryoplasty (p<0.001) and MCP-1 levels were higher after cryoplasty compared with angioplasty and angiography (repeated Anova: p = 0.086).
TGF
Serum levels of TGF dropped in patients with both angioplasty and cryoplasty at 60 min after the intervention and increased afterwards to return close to their baseline levels at 4 weeks after intervention. Again no significant difference in TGF levels was seen between cryoplasty and angioplasty over the 4 week period after intervention (repeated Anova: p = 0.224).
Haemolysis parameters during cryoplasty
LDH values obtained before cryoplasty, during cryoplasty and 5 min after cryoplasty are shown in Figure 1. Blood LDH levels were significantly increased in the samples obtained from the segment proximal to the inflated cryoballoon (where the blood flow was temporarily interrupted by the inflated balloon) 15 s after inflation and cooling of the cryoballoon compared with the LDH values before cryoplasty (Anova: p<0.001). LDH levels returned to their baseline levels at 5 min after cryoplasty when the blood flow was restored. No significant changes were observed in the concentrations of the screened electrolytes.
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Discussion |
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The use of drug eluting stents (DES) seems to improve the patency rate but long-term results are still missing and the published data suggest that the results in peripheral arteries are less promising compared with the effects of DES in the coronaries [14, 15].
Both balloon and stent angioplasty overstretch the entire vessel wall and lead to disruption not only of the occluding plaque, but also of the endothelium, the internal elastic lamina and the media. The induced vessel trauma leads to a complex cascade of vessel inflammation characterized by monocyte infiltration of the intima and media, smooth muscle cell (SMC) migration, SMC proliferation as well as an increased extracellular matrix (ECM) production. These pathways are mediated by adhesion molecules, growth factors and cytokines. Increased expression of adhesion molecules such as e-selectin, p-selectin, ICAM, VCAM and MCP-1 leads to migration of monocytes into the vessel wall [16]. Growth factors such as bFGF and TGF mediate increased proliferation of SMCs and intimal cells as well as ECM production [17, 18]. TNF is also involved in the activation of SMCs and their subsequent migration into the intima after denuding injury caused by an angioplasty balloon [5].
Several studies also proved that the soluble forms of these markers detected in the serum are indicative of endothelial damage and vessel inflammation and that some of these markers are even predictive for the development of restenosis [9, 19].
Therapeutical freezing is now proposed as a new method in the prevention of restenosis especially after peripheral PTA [20]. Apoptosis induction by application of cold shocks has been studied extensively and cryosurgery has been widely used to treat tumours and other diseased tissues characterized by high cellular proliferation rates [21, 22]. Cryoplasty has been suggested as a new method inhibiting restenosis development after interventional recanalization [23]. The cryothermal energy that is distributed to the target lesion while dilating the vessel should damage the adjacent intima cells to a degree large enough for apoptosis induction but without immediate necrosis of the treated area. Apoptosis of injured endothelial cells despite their proliferation may lead to reduced inflammation of the dilated segment and decreased neointimal proliferation. This decreased degree of vascular inflammation and reduced proliferation rates might be reflected by reduced levels of adhesion molecules, growth factors and cytokines.
However we did not find clear evidence that cryoplasty leads to a different degree of vessel inflammation compared with balloon angioplasty. Levels of the adhesion molecules (e-selectin, p-selectin, ICAM, VCAM, MCP-1), the growth factors (bFGF and TGF) as well as the cytokine TNF only differed moderately between angioplasty and cryoplasty.
TNF and bFGF could not be detected in both groups. The extent of local damage to the vessel wall induced by the angioplasty procedure may be too small to induce a detectable difference in the blood levels of these factors. This observation is in accordance with other serologic studies that did not detect TNF changes in human PAOD [24]. The absence of an increased TNF after cryoplasty does not support the theory of an increased apoptosis rate in cryoplasty vs angioplasty.
An increase in soluble bFGF was found in patients with stable angina after percutaneous transluminal coronary angioplasty (PTCA) [25]. The absence of bFGF after angioplasty or cryoplasty in PAOD may be due to the minor capacity of endothelial cells and medial SMCs to release bFGF compared with ischaemic myocardial cells which are able to secret large amounts of bFGF to promote collateral vessel growth in the ischaemic myocardium.
Both procedures lead to increased release of ICAM, VCAM and MCP-1 reflecting a higher degree of endothelial activation and inflammation but the rise is not significantly different between angioplasty and cryoplasty. Only e-selectin levels were significantly higher in the cryoplasty group than in the angioplasty group. But since the baseline e-selectin serum levels were already elevated in the cryoplasty group, the higher levels do not reflect an altered endothelial response compared with the angioplasty group. The reduced p-selectin levels in the cryoplasty group at 4 weeks compared with the significantly elevated levels in the angioplasty group might suggest a benefit of cryo- compared with angioplasty. But since only p-selectin shows decreased levels in comparison with angioplasty at one time point while the other screened biomarkers did not show a significantly reduced expression in the cryoplasty group, a definite superiority of cryoplasty cannot be claimed.
Since the cooled cryoplasty balloon not only has direct contact with the adjacent vessel wall but also with the interrupted blood stream, it is not surprising that haemolysis reflected by increased LDH levels occurs in the segment proximal to the cryoplasty balloon while it is being cooled and inflated. However this haemolysis remains a local phenomenon and disappears without systemic consequences after the blood flow has been restored.
In conclusion, the present study shows: (a) both angioplasty and cryoplasty lead to increased levels of e-selectin, ICAM, VCAM and MCP-1 which reflects altered endothelial activation and inflammation, (b) that the humoral response to cryoplasty in our study was similar to balloon angioplasty, (c) cryoplasty causes haemolysis in the arterial segment proximal to the inflated and cooled balloon which remains a local phenomenon that resolves quickly after the blood flow has been restored.
There are some limitations of the study. The investigated biomarkers are not specific markers for vessel inflammation and are elevated in several diseases with a high degree of inflammation such as neoplastic diseases. Although we tried to minimize the influence of other contributing pathophysiology on the marker levels by excluding patients with known malignant diseases or vasculitis, there is no proof that the elevated levels of the investigated biomarkers derive only from vessel inflammation. The different baseline values of the marker levels between the angioplasty and cryoplasty groups represent a certain confounding bias which has to be taken into account in the interpretation of the data. Further studies are necessary to validate the proposed biomarkers as well as to search for new markers that are highly specific and representative for changes in the arterial biology. A second limitation is the fact that we did not differentiate the marker levels between stenotic lesions and occlusions due to the small sample size. There may be differences in the expression of biomarkers in stenotic vessels and occluded vessels and larger studies are necessary to investigate this possible difference. Another limitation of the study is the small sample size of 29 patients who were not randomized with the potential for a bias for non-randomized allocations. Larger randomized studies with an extended observation period will be necessary to prove and clarify the role of biomarkers in the evaluation of vessel inflammation. Also a differentiation of the biomarker levels between patients with only intermittent claudication and patients with critical limb ischaemia might be helpful.
The results of our study together with the recent published clinical trials that showed little if any benefit of cryoplasty over angioplasty make it questionable whether the apoptosis theory suggested by animal models and in vitro studies is applicable to the clinical use of cryoplasty in human vessels [3, 23, 26–28].
Further studies investigating the in vivo expression of adhesion molecules, growth factors and cytokines responsible for restenosis development are necessary to prove if cryoplasty really leads to an increased apoptosis and a decreased inflammation rate of the treated segment in human arteries.
The study was funded by the Department of Vascular Surgery, Technical University of Munich. The authors declare no financial conflicts.
Received for publication June 26, 2006. Revision received August 18, 2006. Accepted for publication September 5, 2006.
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TopAbstractIntroductionMethods and materialsResultsDiscussionReferences |
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