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Cardiovascular Research 1999 41(2):433-442; doi:10.1016/S0008-6363(98)00211-9
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Copyright © 1999, European Society of Cardiology

Stents covered by an autologous arterial graft in porcine coronary arteries: feasibility, vascular injury and effect on neointimal hyperplasia

Christodoulos Stefanadisa,*, Konstantinos Toutouzasa, Eleftherios Tsiamisa, Charalambos Vlachopoulosa, Sophia Vainaa, Dorothea Tsekouraa, Lubna Haldia, Elli Stefanadia, Michael Gravanisb and Pavlos Toutouzasa

aDepartment of Cardiology, University of Athens, Athens, Greece
bDepartment of Pathology, Emory University, Atlanta, GA, USA

* Corresponding author. Tel.: +30-1-671-8694; fax: +30-1-645-7230.

Received 16 January 1998; accepted 14 March 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The use of stents has improved results after balloon coronary angioplasty. Several materials have been proposed for covering the metallic surface of the stent to reduce the rate of subacute thrombosis and restenosis. In our institution, an autologous arterial graft was used for covering the external surface of a conventional stent. The angiographic and histological response in a porcine coronary artery model was investigated. Methods: An autologous arterial graft was removed from the femoral artery and carefully prepared. Subsequently, a conventional stent was covered externally by the arterial graft. Twenty-two covered stents and 22 uncovered regular stents were implanted alternatively in the coronary arteries of 22 pigs. One animal died immediately after the procedure, due to thrombus formation in the uncovered stent. Six animals were sacrificed at seven days and the remaining animals were sacrificed at two months. Before the sacrifice, coronary angiography was performed in all animals. Results: Thrombosis was detected in two control segments and in one covered stented segment. After seven days, the luminal surface of the covered stents was covered by a new endothelial layer in contrast to partial endothelial cell appearance in the control group. The angiographic parameters were similar between the two groups. Histologically, the covered stents were associated with less vascular injury compared to uncovered stents. In covered stents a trend towards reduction of maximal intimal hyperplasia was detected (covered: 116.6±47.75 vs uncovered: 150.25±46.81 µm, p=0.08); also the thickness of the arterial media was reduced (covered: 21.34±10.28 vs uncovered: 102.63±18.71 µm, p=0.02). The luminal and vessel areas were similar in the two groups. Conclusions: The preparation and implantation of the autologous arterial graft-covered stent is technically safe and feasible. This type of covered stent results in accelerated endothelialization, less vascular injury, thinning of the arterial media and a trend to reduce the intimal hyperplasia in normal coronary arteries.

KEYWORDS Vasculature; Arteries; Histopathology; Restenosis; Stents; Thrombosis


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Clinical trials demonstrated beneficial effects of stents compared to balloon angioplasty [1–3]. However, thrombogenicity and pronounced intimal hyperplasia observed after stenting still represent considerable challenges. The main factors contributing to these unfavorable results of conventional stenting are the delayed endothelialization procedure, the limited biocompatibility of the metallic stents, and the significant vascular injury during the deployment of stents [1–9]. In an effort to address these limitations of stenting, several types of covered stents have been proposed to improve the quality of stents and are currently under investigation [5–12].

In our institution, the experimental and clinical application of a stent covered by an autologous venous graft yielded encouraging results [10, 11]. Furthermore, other investigators have reported their clinical experience in several indications in human coronary arteries [13, 14]. Several studies have revealed favorable results of arterial grafts used as free bypass grafts compared to saphenous vein grafts regarding long-term patency [15–17]. Concerning these superior results of arterial bypass grafts, a new type of stent coverage, an autologous arterial graft, was used. We report the results after the implantation of the autologous arterial graft-covered stent (AAGCS) in normal porcine coronary arteries. The purposes of this study were (1) to determine the feasibility and safety of AAGCS preparation and percutaneous implantation, and (2) to compare the short- and mid-term angiographic patency, and the histologic response of the arterial wall after AAGCS implantation in comparison with those after conventional stenting.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Experimental protocol
Twenty-two domestic normocholesterolemic swines were studied (weight 20–25 kilograms), in which 22 AAGCSs and 22 conventional stents (control group), were placed in alternate order in the left anterior descending (LAD) and left circumflex coronary artery (LCx). The study was approved by the review committee of our institution. All animals were cared, premedicated, anesthesized, and monitored as previously described [11]. The investigation conforms with the Guide for the Care and Use of Laboratory Animals.

2.2 Arterial graft harvest and coverage procedure
Under sterile conditions, an 8F introducer sheath was advanced into the right femoral artery by surgical cutdown. The femoral artery was subsequently exposed in a length of 4–6 cm distal to the arterial cutdown and a 3–4 cm-long arterial graft was removed from this exposed segment of the femoral artery. The diameter of the harvested arterial graft was 4–6 mm. The femoral artery was then ligated distally. Thereafter, the arterial graft was cleared from the surrounding adipose tissue and trimmed to approximately 15 mm length. During the procedure of arterial graft preparation, an angiography of the left coronary artery was performed for the selection of the appropriate balloon-catheter. A pre-mounted Multi-link stent of 15 mm in length (Advanced Cardiovascular Systems, Hampshire, UK) was then selected and the arterial graft was manually placed on the external surface of the unexpanded stent. Finally, three sutures (Prolene 7-0, Ethicon, Edinburgh, UK) were applied at each site of the stent to stabilize the arterial graft on the struts of the stent with care to avoid rupture of the balloon (Fig. 1).


Figure 1
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  		Fig. 1 Schematic representation of AAGCS preparation. An arterial graft of appropriate size is removed and further prepared carefully so as to attain a very small thickness (A). After preparation, the arterial graft is placed on the external surface of the stent (B). Then, the endings of the arterial graft are sutured at the struts of the stent (C). AAGCS indicates autologous arterial graft-covered stent.

 
2.3 In vitro test
After AAGCS preparation, in vitro testing was performed. Seven AAGCSs were prepared and several attempts were performed in order to select the appropriate guiding catheter. An 8F guiding catheter was adequate for the comfortable advancement of all AAGCSs. Subsequently, the AAGCSs were deployed and they were examined macroscopically.

2.4 Stent implantation
An 8F hockey-stick guiding catheter was positioned in the left coronary ostium and coronary angiography was performed. The internal diameter of the proximal segment of the LAD and the LCx was then calculated after calibration, based on the known internal diameter of the angiographic catheter (Image Vue Workstation, Nova Microsonics). The model of overstretch injury has been previously described [18]. One of the LAD or the LCx arteries in each swine was assigned for the implantation of an AAGCS or an uncovered stent (15-mm in length, Advanced Cardiovascular Systems, Hampshire, UK) in alternative order. Intentionally in both types of stents, an overexpansion of the balloon was performed by a 1.2 ratio to the normal coronary lumen in order to produce the characteristic vascular injury (Fig. 2) [18]. Repeat angiography was then performed to confirm adequate stent expansion and vessel patency. The oversizing ratio was calculated in both groups by subdividing the maximal balloon diameter to the diameter of the stented vessel segment. No further anticoagulant or antiplatelet treatment was given to the animals. One animal died immediately after the end of the procedure. The animals were maintained on a standard laboratory chow diet throughout the study period.


Figure 2
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  		Fig. 2 Schematic representation of AAGCS implantation. The covered stent is delivered at the target vessel (panel A), the balloon is then deployed (panel B), and it is positioned at the arterial wall (panel C).

 
2.5 Follow-up examination
Arterial access was obtained by left femoral artery cutdown. Follow-up angiography was performed seven days after stent implantation in six animals and at two months in the remaining 15 animals. The minimal luminal diameter (MLD) was calculated as previously described (see ‘Methods', ‘Stent implantation'). The following parameters were also calculated: Immediate gain (MLD after AAGCS implantation – luminal diameter before AAGCS implantation) and late loss (100x[MLD at follow-up MLD after the implantation]/MLD after the implantation,%).

2.6 Tissue processing
Immediately after the follow-up angiography, the animals were sacrificed. Thus, the first six animals were sacrificed seven days after stent implantation and the remaining 15 animals after two months. Sacrifice was performed in accordance with Animal Welfare Regulations outlined by the American Physiologic Society. The heart was rapidly excised and the coronary system was perfusion-fixed at 110 to 120 mm Hg driving pressure with 10% formaldehyde for 15 min. The stented and the adjacent unstented segments were kept in 4% formaldehyde in phosphate buffer, pH 7.3, for at least 48 h.

In random order, either the distal or the proximal half of the segment was further processed for light microscopy and the other half for scanning electron microscopy observation. The specimens for light microscopy were oriented for distal and proximal ends and they were embedded in methyl methacrylate and sectioned with a tungsten carbide knife. Serial 4-µm sections were taken and morphometric evaluation was performed in every tenth section. Hematoxylin and eosin, van Giesson's elastin, and Masson's trichrome stains were used. All sections were examined by an experienced observer. After deplastization of other unstained sections, an immunohistochemical method was used to detect any proliferating activity. Immunohistochemical staining was performed by peroxidase–antiperoxidase staining procedures (Dako Corp). The anti-proliferating cell nuclear antigen (PCNA) was used (Dako Corp). The adjacent non-stented areas were used for negative control sections and breast tumor tissues for positive sections.

2.7 Assessment of vascular injury
Each specimen was evaluated for the presence of intraluminal thrombus, neointima formation, luminal encroachment, alterations of the internal and external elastic membrane, alteration of the covering arterial graft, and morphologic appearance of the cells within the media, adventitia, and neointima. Also, the vessels were graded on a scale to assess the degree of vascular injury (I) as follows: I1 for no disruption of the internal elastic membrane (IEM); I2 for penetration of at least one strut into the arterial media; I3 for disruption of the external elastic membrane [18–21].

2.8 Scanning electron microscopy
The specimens for scanning electron microscopy were washed with PBS, placed in osmium tetroxide and washed again with PBS. Thereafter, tissues were dehydrated using ethanol and dried at critical point drying. Finally, tissues were covered with gold and palladium. The stented segment was cut longitudinally, opened along the longitudinal cut, and examined under a scanning electron microscope. All specimens were examined for the presence of thrombus, luminal encroachment, and degree of endothelialization. The area of endothelial denudation in the stented segments was quantified by computer-assisted digital planimetry [22]. The quantitative analysis was confined to the continuous longitudinal section of the endoluminal surface that remained flat after the longitudinal cut of the specimens.

2.9 Morphometric studies
By using computer-assisted digital planimetry (Cue-2, Olympus) the following parameters were measured: luminal area (LA), maximal intimal thickness (MIT, defined as the maximal distance from the IEM to the lumen, without including the autologous arterial graft in AAGCS sections), and thickness of the arterial media of the artery (M, defined as the maximal distance from the external elastic membrane to the IEM, measured at the sites between the stent wires). The measurements were performed by two observers and in at least five sections of each stented segment.

2.10 Statistical analysis
Values are expressed as mean±SD. For comparison of parameters between the AAGCS segments and the control segments, paired t-test was used. Vascular injury score was compared by the Mann-Whitney U test. Statistical significance was set at p<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 AAGCS preparation, in vitro test and implantation
In all cases, the process of covering the stent with an autologous arterial graft was feasible and easy. The in vitro deployment of seven AAGCSs showed that the arterial graft completely covered the external surface of the stent and followed the expansion of the stent. Macroscopically, the in vitro deployment was performed without any complication and neither flapping of the arterial graft nor rupture of the delivery balloon was observed. The in vivo delivery of the AAGCS in the porcine coronary arteries was successful in all cases. Also, during the implantation of the AAGCS, no complication was detected.

3.2 Angiographic results
Eleven uncovered stents were implanted in the LAD and 11 in the LCx. Eleven AAGCSs were placed in the LAD and 11 in the LCx. Deployment of the stents was successful in all cases, as evidenced by immediate angiography. The AAGCS was stabilized on the wall of the artery and did not migrate from its original position. There was no case of rupture, dissection, or peripheral embolization of the coronary arteries. The oversizing ratio was similar between the two groups (AAGCS segments: 1.19±0.15; control: 1.17±0.15, p=0.31). Acute thrombosis was observed in two control segments immediately after implantation. In the first segment a repeat prolonged dilatation was performed, achieving an acceptable angiographic result. This animal, however, died immediately after the end of the procedure. The other animal survived but a thrombus was detected at seven days. The stented segment was redilated and the immediate angiography revealed patency of the vessel. The animal was then purposefully sacrificed. All other segments of both groups remained patent at the seven-day angiography. The immediate gain was similar between the two groups. Also, the luminal diameter at seven days was similar between AAGCS and control segments (2.71±0.57 mm vs 2.77±0.56 mm, n=6, p=0.25). The angiographic parameters of the two groups at two months had no statistical difference (Fig. 3, Table 1).


Figure 3
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  		Fig. 3 Angiography of the coronary arteries of an animal two months after the procedure. The non-covered stent was implanted in the LAD (A) and the AAGCS in the LCx (B). After two months, the vessels are patent (C). Arrows indicate the stented segments. AAGCS indicates autologous arterial graft-covered stent; LAD, left anterior descending; LCx, left circumflex.

 

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  		Table 1 Angiographic results

 
3.3 Light microscopy
The autologous arterial graft was detectable in all AAGCS sections. The covering material was thinner than the thickness of the native arterial wall, due to the preparation of the arterial graft before the procedure of stent covering. At seven days, an organized thrombus was detected in one AAGCS and one uncovered stented segment. After two months the luminal surface of all AAGCS segments was covered by a mature endothelium providing a smooth surface. In the uncovered stents, compression of the media was detected at the sites of the stent wires. In several sites, the internal elastic membrane was injured and the arterial media was lacerated. In both groups, occasional ridges in the luminal surface consisting of smooth muscle cells and extracellular connective tissue matrix were revealed. In AAGCS sections, a mild smooth muscle cell proliferation along with extracellular matrix was observed in the neointimal layer, especially at the areas beneath the stent wires. PCNA staining demonstrated a minimal amount of positive cells in the neointimal layer and in the autologous arterial graft, indicating that viable cells exist. A new elastic membrane between the neointimal layer and the arterial covering graft was detected in all AAGCS sections. Although the covering arterial graft was compressed at the sites of the stent struts, the stent wires did not penetrate the covering arterial graft in any section. The metallic surface of the stent was not adjacent with the arterial media, and thus the internal elastic membrane and the arterial media were intact. Moreover, a thinning of the arterial media was observed across the vessel circumference. In addition, no inflammatory response was detected across the arterial covering demonstrating the biocompatibility of the endoprosthesis (Figs. 4 and 5Go).


Figure 4
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  		Fig. 4 Light microscope images of control (A) and AAGCS (B) segments, two months after the procedure (hematoxylin–eosin stain). The arterial graft (A) is detected between the I and the M of the native arterial wall. AAGCS indicates autologous arterial graft-covered stent; I, neointima; M, media; A, arterial graft.

 

Figure 5
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  		Fig. 5 Light microscope image (A) two months after uncovered stent implantation (van Giesson's stain) showing the laceration of the internal elastic membrane. In panel B, a new membrane (new IEM) differentiating the arterial graft from the intimal layer is revealed, resembling the IEM of the native media. The IEM of the native artery is intact. Additionally, the thickness of the media is reduced and a minimal intimal hyperplasia is observed. A schematic representation (panel C) of the layers of the arterial wall in AAGCS section. AAGCS indicates autologous arterial graft-covered stent; IEM; internal elastic membrane; EEM, external elastic membrane.

 
3.4 Vascular injury
An intentional overexpansion of both types of stents was performed. However, the oversizing ratio was similar in both groups. Histological grading of the stented segments showed significant differences between the covered and uncovered stents at seven days and two months examination (Table 2). The uncovered stent was associated with higher grades of vessel injury. The arterial covering was intact without fragmentation or disruption. All AAGCS segments were graded I1 (no disruption of the IEM) except 1 case in which fragmentation of the IEM was revealed (I2). In the majority of the uncovered stented sections, fragmentation of the IEM with transection of the arterial media from the stent struts was observed (I3) (Table 2).


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  		Table 2 Injury variables of porcine coronary arteries

 
3.5 Scanning electron microscopy
Seven days after the procedure a new endothelial layer was detected across the luminal surface of the AAGCS specimens covering also the stent struts. The luminal surface has relined with regenerated endothelium, which was flow-oriented. In contrast, some areas of the luminal surface of the control segments showed endothelial cell loss. In AAGCS sections 81±12% of the luminal surface was covered by the endothelial cells, in contrast to 52±14% of the control group (p<0.03). Additionally, denuded smooth muscle cells at the luminal surface of the uncovered stents were presented (Fig. 6). In both stents, the endothelium displayed a mixture of polygonal and elongated morphology with occasional leukocyte adherence. Also, at two months all stents were incorporated into the vessel wall. Complete endothelial coverage was observed in both groups. No rupture of the autologous arterial graft was detected. All specimens showed a smooth luminal surface without evidence of attached microthrombi or platelets.


Figure 6
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  		Fig. 6 Scanning electron microscope images (x600) of the luminal surfaces of control (A) and AAGCS (B) segments seven days after the implantation. In both types of stents, a partial coverage of the luminal surface by endothelial cells is observed. However, the surface of the AAGCS segment is smoother, possibly due to the presence of the arterial graft in contrast to denuded areas in the luminal surface of control segments (arrows in panel A). AAGCS indicates autologous arterial graft-covered stent.

 
3.6 Morphometric studies
The results of the morphometric studies are shown in Table 3. The mean luminal area was similar between the two groups, two months after the implantation. The mean maximal intimal thickness was greater in the control group compared with AAGCS segments although the difference did not reach statistical significance (p=0.08). The mean thickness of the arterial media was less in the AAGCS segments compared with conventionally stented segments (p=0.02).


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  		Table 3 Morphometric results at two months

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The results of this study showed that the procedure of AAGCS implantation in porcine coronary arteries is feasible and safe. This type of covered stent provides biocompatibility since inflammatory response was not detected. The implantation of AAGCS seems to have favorable effects on the rate of endothelializaton and the extent of vascular injury. During the follow-up period, a minimal hyperplasia appeared and it was associated with thinning of the arterial media.

4.1 Rationale for autologous arterial graft covering
Despite the established benefits of stent implantation, several limitations remain to be addressed [2–4]. Subacute thrombosis and restenosis still are the targets of current research. Improving the surface characteristics of stents may further enhance the clinical results of coronary stenting by eliminating factors of thrombosis and intimal hyperplasia. In an attempt to solve these problems while retaining the benefits of stenting, covered stents have been proposed. Covering of stents aims at providing biocompatibility, accelerating the endothelialization procedure, minimizing the vascular injury and reducing the proliferative response of the native arterial wall. Several agents have been utilized as sleeves for the stents, both experimentally and in clinical practice [5–9]. Of them, only heparin-coated stents had favorable results in clinical application [5, 6]. Previous studies from our institution have shown favorable experimental and clinical results after the implantation of stents covered by venous grafts. Clinical studies with arterial grafts used as free bypass grafts showed favorable results compared to saphenous vein grafts in terms of long-term patency [15–17]. Based on these superior results, a new type of stent coverage, an autologous arterial graft, was used. In the present study, we investigated the angiographic and histological outcome in porcine coronary arteries after the implantation of the AAGCS.

4.2 Effect of arterial graft covering on factors of thrombosis
The procedure of endothelialization is a significant antithrombotic factor, which may be more important in the presence of an atherosclerotic plaque. Occlusion of a stent by thrombus occurs primarily during the first week after implantation. In uncovered stents, endothelial cell coverage was completed after 4 weeks in rabbit carotid arteries [20]. The results from scanning electron microscopy demonstrate an accelerated endothelialization in AAGCS segments, seven days after the procedure. Thus, covering the stents by an autologous vascular graft, either externally or completely, seems to enhance the formation of the new endothelial layer.

The application of an autologous arterial graft on the surface of the stent provides biocompatibility. In addition, it sequestrates the atherosclerotic plaque and prevents the contact between the atherosclerotic plaque and the bloodstream. The implantation of AAGCS prevents the protrusion of the atheromatic tissue into the lumen. Moreover, by the sequestration of the atherosclerotic plaque a smooth luminal surface is provided in contrast to the rough surface of conventional stented segments. This may be important in thrombus-containing or severely diseased lesions in which the atherosclerotic plaque may prolapse through the stent struts or articulation sites [12].

4.3 Effect of arterial graft covering on factors of neointimal hyperplasia
Although the angiographic results were similar between the AAGCS and control group, in covered stents a trend to reduce the maximal intimal hyperplasia was detected by morphometry. Both covered and non-covered stents had similar oversizing ratios. However, the arterial wall expansion during stent implantation was greater in AAGCS group due to the thickness of the arterial graft. Thus, by using the same balloon diameter, the arterial wall was expanded more in the AAGCS segments in order to achieve a similar luminal diameter to the control group. However, AAGCS was accompanied by less injury score and minimal intimal hyperplasia. Previous studies have shown that the integrity of the internal elastic membrane is essential to minimize neointimal proliferation [19]. The autologous arterial graft prohibits the contact between the struts of the stent and the internal elastic membrane. The autologous arterial graft, however, is not solely an anatomical barrier, but also contains viable cells. Although, the origin of these cells is unknown, the results from the PCNA staining show that the autologous arterial graft has functional properties.

Morphometric analysis revealed that the thickness of the native arterial media of the AAGCS segments was thinner compared to the control group. Media atrophy has previously been reported only at the sites of the stent wires and it was correlated with arterial media injury [21]. In the presence of arterial covering, the underlying mechanism seems to be different, since the injury of the native arterial media was minimal. Therefore, this observation may be ascribed to the reduced wall stress due to the existence of the arterial graft. Moreover, the arterial graft may impair the nutrition from the lumen towards the arterial wall and may lead to a compromise of nutrients diffusion leading to medial atrophy [23, 24].

4.4 Methodological considerations
The autologous arterial graft was appropriate for the diameter of the porcine coronary arteries. Also, the covering and implantation procedure did not induce any damage of the arterial graft, as was confirmed by in vitro studies, and scanning electron and light microscopy observation during the follow-up.

Stent thrombosis appears to be modulated by several mechanisms including incomplete stent expansion, vascular injury, non-biocompatibility of the device, and systemic factors such as the effectiveness of platelet or thrombin inhibition [25]. These thrombogenic factors may be eliminated by the arterial covering, except for the incomplete stent expansion. On this occasion, the struts of the stent may prolapse into the lumen and cause thrombosis. Covering the stent both internally and externally by an autologous vascular graft may protect from thrombus formation in such cases. However, the thickness of the arterial graft is prohibitive for complete coverage of the stent. A possible solution to this problem may be the reduction of the arterial wall thickness and thus the stent would be completely covered.

The delivery of the AAGCS to the porcine coronary arteries was uncomplicated. However, the AAGCS may not be implanted in vessels <2.5-mm due to the increased profile of the device. These results are in accordance with the clinical experience, in which no complication was observed during the preparation of the AAGCS. In contrast, occlusion of arterial side branches at the target lesion after AAGCS delivery appears to be a limitation of this method. A limitation of the study appears to be that the sections could not be blindly assessed, due to the existence of the arterial graft. However, the implantation of both types of stents was randomly performed.

4.5 Clinical implications
The preliminary experience with AAGCS in human coronary arteries showed that it is a feasible procedure. In order to apply the experimental results in the clinical practice [26], a graft from the radial artery was used for covering a conventional stent [12]. Although the invasiveness of the procedure is increased, no complication was observed. Also, surgeons successfully used the radial artery as a free graft for coronary artery by-pass surgery. The implantation of covered stents by an autologous arterial graft may sequestrate the atherosclerotic plaque and especially in diffuse coronary artery disease or in thrombus-containing lesions. Indeed, the preliminary results after the implantation of the covered stents by autologous arterial grafts in human coronary arteries are encouraging, especially in thrombus-containing lesions. The procedure of AAGCS preparation is not prolonged and it also may be used as an emergency procedure in complications of currently used invasive techniques. The AAGCS may also be used for reconstruction of disruptions of vessel wall integrity, such as dissections, perforations, and aneurysms [13, 14].

4.6 Summary
Covering the stent by an autologous arterial graft may help in eliminating the limitations of stenting and may appear as an appealing treatment for coronary artery disease. However, the encouraging results after the experimental application of the autologous arterial graft-covered stent must be confirmed by large clinical trials, which will define the efficacy of this approach.

Time for primary review 28 days.


   Acknowledgements
 
This investigation was supported by a grant from the Hellenic Heart Foundation and the Hellenic Cardiological Society.


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

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