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Cardiovascular Research 2002 53(2):502-511; doi:10.1016/S0008-6363(01)00486-2
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Copyright © 2001, European Society of Cardiology

Endothelial cell seeding influences lesion development following arterial injury in the cholesterol-fed rabbit

Michael S Contea,d,*, Guy A VanMetera,d, Lee M Aksta, Tracy Clemonse, Michael Kashgarianc and Jeffrey R Benderb,d

aDepartment of Surgery, Yale University School of Medicine, New Haven, CT, USA
bDepartment of Medicine (Cardiology), Yale University School of Medicine, New Haven, CT, USA
cDepartment of Pathology, Yale University School of Medicine, New Haven, CT, USA
dThe Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT, USA
eHarvard University School of Public Health, Boston, MA, USA

* Corresponding author. Department of Surgery, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, USA. Tel.: +1-617-732-6816; fax: +1-617-730-2876 mconte{at}partners.org

Received 16 March 2001; accepted 1 October 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: A functionally intact endothelial monolayer is thought to be critical for the adaptive process of vascular remodeling. This study was undertaken to examine the hypothesis that endothelial restoration is a critical determinant of remodeling after balloon angioplasty. Methods: Rabbits (N=12) were fed a cholesterol-supplemented diet (0.5%) and were subjected to bilateral balloon catheter injury of the iliofemoral arteries. At the time of injury, autologous venous endothelial cells (ECs) were implanted on one artery; the contralateral vessel served as control. A mean of 42 days after injury, arteriography was performed, followed by vessel harvest and histologic analysis. Results: High grade (≥70%) stenoses or occlusion were present in 55% of control and none of the EC-seeded arteries. EC-seeding was associated with improved mean (1.0±0 vs. 0.7±0.1, P<0.001) and minimal (0.7±0.1 vs. 0.4±0.1, P<0.001) luminal diameters by angiography. Seeded arteries demonstrated decreased medial area (0.69±0.04 vs. 1.04±0.09 mm2, P<0.001), a more uniform range of final lumen area (P<0.0001), and a positive remodeling index. Neointimal area was not significantly different. Stenoses were characterized primarily by larger neointimal area (2.02±0.18 vs. 1.38±0.09 mm2, stenotic vs. non-stenotic, P<0.005). Final lumen area was strongly influenced by both neointimal growth and vessel remodeling. Conclusions: These data support the concept that endothelial restoration is a critical determinant of the outcome of vessel wall repair, particularly in the context of hypercholesterolemia.

KEYWORDS Angioplasty; Arteries; Endothelial function; Restenosis; Cholesterol


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Restenosis significantly limits the durability of all types of reconstructions performed on small to medium-sized arteries in the coronary, cerebral, and peripheral circulations. The pathologic hallmark of restenosis, intimal hyperplasia (IH), is a flow-restricting fibrocellular lesion composed primarily of vascular smooth muscle cells (SMCs) and extracellular matrix. SMC migration, proliferation, phenotypic alteration, and matrix secretion are important processes involved in this characteristic response to vascular injury. Recent studies have suggested that recurrent luminal narrowing (e.g., following balloon angioplasty) is a complex phenomenon that is not related solely to the magnitude of IH, and have implicated an impaired remodeling response of the healing arterial wall [1–3]. Current approaches to limit restenosis, including endoluminal stents and local radiotherapy, appear to exert a beneficial influence by addressing both the hyperplastic and remodeling aspects of this process.

Substantial evidence supports a critical role for the endothelial cell (EC) monolayer in the structural adaptation of blood vessels (remodeling) that accompanies changes in hemodynamic conditions as well as vessel injury [4–6]. Remodeling may be defined as the process by which overall vessel size changes in response to such stimuli. All vascular reconstructions unavoidably result in endothelial injury, and the endothelium which subsequently regenerates may be at least transiently dysfunctional [7]. An intact EC lining may influence vascular repair by numerous mechanisms including elaboration of locally acting cytokines and vasoactive substances as well as modulation of interactions with blood elements. Previous investigations in animal models of arterial injury have suggested an inverse relationship between the rate and completeness of endothelial regeneration and the extent of IH that develops [8–11]. While the cellular and molecular events involved in post-injury remodeling remain undefined, endothelial integrity is likely to be a critical determinant of the outcome of arterial repair.

We have previously characterized a rabbit arterial injury model in which cultured, autologous, venous-derived ECs are implanted (‘seeded’) on the freshly denuded vessel surface [12]. In the normal chow-fed rabbit, balloon arterial injury results in a modest degree of IH without luminal narrowing, and neointimal lesion formation is not significantly altered by EC seeding [13]. We hypothesized that re-endothelialization might be particularly critical following mechanical injury in an atherogenic environment, where the inflammatory and hyperplastic responses to injury are upregulated.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animal procedures
Adult New Zealand white rabbits (N=12) weighing between 3 and 5 kg were given free access to water and rabbit chow and were housed in a facility with alternating light and dark cycles. The experimental protocol was approved by the Yale Animal Care and Use Committee. Animal care and procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, Washington: National Academy Press, 1996).

Animals were fed a cholesterol-supplemented diet (consisting of normal rabbit chow +0.5% (w/w) cholesterol and 5% (w/w) hydrogenated coconut oil; Research Diets, New Brunswick, NJ). Previous experience with this diet in rabbits had demonstrated rapid progression of arterial lesions to stenosis within 4 to 8 weeks after balloon injury. The diet was maintained for at least 2 weeks prior to arterial injury and thereafter up to sacrifice. Dietary tolerance was 100%. Serum lipid determinations were performed at sacrifice.

Animals were anesthetized for surgical procedures with ketamine (25 mg/kg) and xylazine (5 mg/kg) administered by intramuscular injection, supplemented with intravenous doses of the mixture as needed. Jugular veins were harvested as previously described [12]. A 10 ml sample of blood was drawn for autologous serum. The neck incision was closed with absorbable sutures and the animal recovered.

2.2 Endothelial cell isolation and culture
The freshly excised jugular vein segment was rinsed with Medium-199 (M-199, Gibco-BRL, Grand Island, NY), then filled with 0.2% Type I collagenase (Worthington, Malvern, PA) in M-199 supplemented with 1% fetal bovine serum (FBS, Gibco). The filled vein segment was incubated in a water bath at 37°C for 10 min. Under a laminar flow hood, the collagenase effluent was collected together with several milliliters of complete EC medium [M-199 with 20% (vol/vol) FBS, 17.5 units/ml of porcine intestinal mucosa-derived heparin (Sigma, St. Louis, MO), 100 µg/ml of endothelial cell mitogen (Biomedical Technologies, Stoughton, MA), 0.7 mmol/l of L-glutamine (Gibco), 50 units/ml of penicillin G sodium, 50 µg/ml of streptomycin sulfate, and 125 ng/ml of amphotericin B (Antibiotic–antimycotic, Gibco)] which was forcefully flushed through the vein. Cells were pelleted (125xg for 10 min), then resuspended in complete EC medium and plated to tissue culture dishes precoated with gelatin (1% gelatin in 0.9% saline, Difco Laboratories, Detroit, MI). ECs were cultured at 37°C in a humidified, 5% carbon dioxide atmosphere. Culture medium was changed routinely three times per week and the cells allowed to proliferate to near confluence prior to passage, typically at 7–10 days after harvest. Cells were passaged with 0.05% trypsin–EDTA (Gibco) and replated to gelatin-coated dishes at ratios between 1:4 and 1:6. Cultures were allowed to grow until sufficient cells were available for seeding [approximately 3x106 cells, which required a mean of 18 (range 12–33) days in culture and maximum of four passages]. EC cultures were characterized using established cytochemical and immunohistochemical techniques [12,13].

2.3 Arterial injury/EC seeding
When an adequate quantity of autologous ECs were available for seeding (mean=18 days) the rabbits were re-anesthetized for arterial injury. The iliofemoral arterial segment was exposed bilaterally from the common iliac bifurcation to the superficial femoral artery (SFA). All branches distal to the origin of the external iliac artery and proximal to the femoral bifurcation (typically 2–4 in number) were ligated, creating an isolated segment for seeding. The SFA was cannulated and a 2-French balloon embolectomy catheter inserted. The catheter tip was passed into the terminal aorta and the balloon inflated and withdrawn three times to denude the vessel. On the third passage a microvascular clamp was placed at the origin of the external iliac artery; the balloon catheter was then replaced by a cannula, and the denuded vessel segment rinsed with M-199 supplemented with heparin (20 units/ml). After directing flush solution down the profunda femoral (PFA) branch, the PFA was clamped at its origin.

Concurrent with the surgical exposure a suspension of autologous cultured ECs was prepared for seeding. Newly confluent, early passage autologous rabbit ECs were detached by trypsinization for 3–5 min at 37°C, and the trypsin inactivated with an equal volume of M-199 supplemented with 20% autologous rabbit serum and heparin (20 units/ml). An aliquot was counted, and the volume adjusted to create a cell suspension of 3x106 cells/ml (calculated to yield approximately 2x105 cells/cm2 of vessel surface). In previous experiments we have demonstrated rapid repopulation of the vessel surface by seeded cells when such initial densities are employed [12]. After flushing the freshly denuded vessel as described, the endothelial cell suspension was infused to gently distend the vessel and left undisturbed for 30 min. The contralateral vessel in each animal was treated identically except for the absence of cells in the infused solution. After incubation, the catheter and clamps were removed, the SFA was ligated, and anterograde flow re-established via the PFA. Wounds were closed surgically and the animals allowed to recover.

2.4 Angiography and tissue harvest
Angiography was performed in nine animals at sacrifice. After inducing anesthesia with ketamine/xylazine the right common carotid artery was exposed and cannulated. A 4-F angiography catheter was advanced under fluoroscopic control (Phillips digital unit) to the abdominal aorta. The animal was sytemically heparinized (1000 units, i.v.). Antero-posterior and oblique projection images of the aortoiliac segment were obtained after injection of 3–5 ml of iodinated contrast (Renograffin-60).

The infrarenal aorta and inferior vena cava were then exposed and cannulated, and the distal arterial tree flushed at a pressure of 100 mmHg with heparinized (10 units/ml) lactated Ringer solution. Animals were sacrificed by intravenous overdose of sodium pentobarbital, followed by perfusion-fixation with PLP (2% paraformaldehyde, 0.075 mol/l lysine, 0.01 mol/l sodium periodate, pH 7.3) for 8–10 min at 100 mmHg. The terminal aorta and distal arterial tree were excised en bloc and stored in fixative at 4°C until processing. Vessels were harvested from two animals solely for scanning electron microscopy (SEM). In these animals, perfusion-fixation was performed using 2.5% glutaraldehyde in 0.1 mol/l sodium cacodylate buffer, pH 7.3.

2.5 Quantitative morphometry
Paired sections (three/vessel, total of 60 sections from 10 animals) were taken at evenly spaced (5 mm) intervals from the proximal, mid, and distal iliofemoral arterial segment in each animal. Paraffin-embedded blocks were sectioned at 6 µm, and stained with Hematoxylin–Eosin and Verhoeff's elastin stain. The cross-sectional areas of the lumen (L), neointima (NI), and media (M) were measured using computer-aided morphometry (NIH Image software). The limits of the neointima were defined by the lumen and the internal elastic lamina (IEL). The media was defined as the area between the IEL and the external elastic lamina (EEL) when visible, or the junction with the adventitia when the EEL was not clearly defined.

2.6 Quantitative angiographic analysis
Angiograms were obtained in nine animals. The projection which most clearly delineated the severity of stenotic lesions was chosen for measurements. Measurements were made by one observer, an interventional cardiologist (JRB), who was blinded to the treatment groups. To account for potential differences in magnification, a reference diameter was recorded from one of the normal-appearing internal iliac arteries on each angiogram. Diameter measurements were then made at 5 mm intervals along the external iliac–common femoral segment, starting 5 mm from the EI origin and ending 5 mm from the origin of the PFA. In addition, the narrowest point of each vessel was recorded as the minimal luminal diameter (MLD) for that side. All diameters were normalized to the reference diameter for statistical analyses.

2.7 Scanning electron microscopy (SEM)
In two animals (21 and 28 days after injury/seeding) the paired iliofemoral arteries were prepared for SEM. Tissue segments were dehydrated in graded concentrations of ethanol, critically point dried with carbon dioxide, bisected longitudinally with a sharp blade and sputter coated with gold/palladium. The samples were observed at 20 kV on an AMR 1400 scanning electron microscope. The surfaces were examined at x100–500 and representative photomicrographs taken.

2.8 Statistical methods
Angiographic diameters and morphometric measurements were compared using a two-tailed t-test (paired for seeded vs. non-seeded arteries, non-paired for stenotic vs. non-stenotic segments). Correlations between compartment measurements were performed utilizing generalized estimating equations (GEE) with linear modeling (SAS software). Statistical significance was based on a P-value <0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
All animals survived the injury/seeding protocol up to scheduled sacrifice (mean 42 days after balloon injury). Serum lipid determinations were performed on eight of the animals. As expected, the diet induced significant elevation of total cholesterol (963±202 mg%) with mild hypertriglyceridemia (176±82 mg%).

3.1 Angiographic results
There was one complete occlusion and four high grade (>70%) stenoses observed by angiography; all of these lesions occurred in non-seeded control vessels. These high grade lesions were segmental, often occurring adjacent to areas of luminal dilation in the same vessel. Statistically significant differences were observed in both mean diameter (1.0±0 vs. 0.7±0.1, P<0.001) and MLD (0.7±0.1 vs. 0.4±0.1, P<0.001) between seeded and non-seeded vessels, respectively. These data are summarized on Table 1; representative angiograms are illustrated in Fig. 1.


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  		Table 1 Summary of angiographic results

 

Figure 1
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  		Fig. 1 Representative angiograms from animals at sacrifice. Under ketamine/xylazine anesthesia, a 4-F angiography catheter was inserted via carotid cannulation and advanced to the abdominal aorta. Following heparinization, images of the aortoiliac segment were obtained after injection of 3–5 ml of iodinated contrast. Tick marks indicate sites of diameter measurements as well as location of reference diameter (uninjured internal iliac). In each case, the right iliac received ECs as treatment and the left served as a non-seeded control. (a) Rabbit sacrificed on day 36. A long, high grade stenosis is seen in the control vessel. (b) Sacrifice on day 46. Tandem high grade lesions seen in the control vessel.

 
3.2 Morphometric analysis
A total of 56 of 60 vessel segments were analyzed; four sections were excluded due to inadequate fixation or artifacts which rendered the anatomic landmarks uninterpretable. To compare results between seeded and non-seeded vessels, compartment areas from the segments were averaged to provide a mean value for each vessel. To gain further insight into the mechanisms of luminal narrowing in this model, we analyzed measurements from the individual segments and compared compartment areas from stenotic (defined by arteriographic narrowing >70%) and non-stenotic regions.

All vessels developed significant neointimal formation as well as medial thickening. Neointimal lesions were composed of lipid-rich (foam cell and extracellular lipid) and fibrocellular (spindle-shaped cells and matrix) regions. Foam-cell rich areas were also observed in the thickened media. No consistent qualitative difference in lesion morphology was observed between seeded and non-seeded arteries.

3.3 Analysis of stenotic sections
Ten histologic sections, all from non-seeded arteries, corresponded to vessel segments identified angiographically as stenoses exceeding 70% diameter reduction (Fig. 2). These were compared to the remaining 46 artery segments (28 seeded, 18 non-seeded) in an attempt to determine the mechanisms of luminal narrowing in this model. Stenoses where characterized primarily by larger neointimal area (2.02±0.18 mm2 vs. 1.38±0.09 mm2, stenotic vs. non-stenotic, P<0.005, Table 2). EEL areas were nearly identical, suggesting a secondary role for remodeling in the progression to luminal narrowing in this model. Injury severity scores (IEL breaks) were not correlated with the development of stenosis.


Figure 2
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  		Fig. 2 Representative histologic sections from a high grade stenosis (x40). Rabbit sacrificed at 42 days after injury. This section was taken from the distal iliofemoral segment on the control (non-seeded) side. A complex lesion is formed in this model with significant neointimal formation and medial thickening. Foam cells as well as extracellular lipid are prominent in the neointima, and, to a lesser degree, the media as well.

 

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  		Table 2 Summary of morphometric analysis of arterial segments

 
3.4 Correlates of lesion severity and role of remodeling
To gain further insight into the relative roles of neointimal growth and remodeling, correlation analyses were performed between the variables L, NI, and EEL. In the pooled analysis of all vessels (seeded and control), both NI (r2=0.13, P<0.01) and EEL (r2=0.09, P<0.05) were weakly correlated with lumen area. A generalized adaptive remodeling response was evidenced by a strong positive correlation between NI area and overall vessel size (EEL=1.715 +0.96 NI, r2=0.6, P<0.0001; all vessels). By multiple regression analysis, the best model for predicting lumen area was: L=–0.17–0.87 (NI)+0.71 (EEL)+0.14 (TRT) (L, NI, EEL in mm2; TRT=1 for EC seeding, 0 for no seeding, r2=0.81).

3.5 Effects of EC seeding on morphometric end-points
The compartment area measurements for control and EC-seeded arteries are summarized in Table 3. Injury severity was not different between seeded and control vessels (82% vs. 74% sections with discontinuous IEL; 2±0.3 vs. 1.7±0.3 IEL breaks). There was no difference in mean lumen or neointimal area, although a trend towards greater neointimal area was noted in the non-seeded vessels (1.38±0.1 vs. 1.62±0.14 mm2, P=0.1). Non-seeded control vessels demonstrated significantly greater medial (1.04±0.09 vs. 0.69±0.04 mm2, P<0.001) and overall vessel wall areas (NI+M: 2.66±0.19 vs. 2.07±0.12 mm2; EEL: 3.49±0.18 vs. 2.91±0.12 mm2, P<0.01).


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  		Table 3 Summary of morphometric analysis of treated vessels

 
EC seeding was associated with a narrower range of lumen areas (P<0.0001, Cochran's test for variances) as compared with control vessels (mean±S.D.: 0.84±0.27 vs. 0.84±0.66 mm2, median: 0.89 vs. 0.66 mm2, EC-seeded versus control, Fig. 3a). Consistent with the angiographic appearance and morphometric data, EC seeded arteries demonstrated a more uniform remodeling response (Fig. 3b, c). The relationship between NI area and overall vessel size (EEL), which may be considered a remodeling index [3], is illustrated for the two groups of vessels on these scatterplots. A slope of >1 indicates positive remodeling, with EEL area accommodating NI growth. A more uniform pattern of remodeling, as evidenced by the r2 values (0.65 seeded vs. 0.39 non-seeded) and 95% confidence intervals for slope (0.72–1.3 seeded vs. 0.4–1.2 non-seeded), was observed in the EC-seeded arteries.


Figure 3
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  		Fig. 3 Scatterplots demonstrating relationships between measured compartment areas from vessel segments (N=56). (a) Plot of lumen area measurements from segments of seeded and non-seeded arteries. Mean values (0.84 mm2) were identical for the two groups; median values are indicated by the horizontal hash mark. Seeded vessels demonstrated a more uniform range of final lumen area (P<0.0001, Cochran's test for variances). (b) Plot of NI versus EEL for seeded arteries demonstrates a strong positive association (r2=0.65, P<0.0001). The slope of 1±0.14 (95% CI 0.7 to 1.3) indicates adaptive remodeling. (c) Plot of NI versus EEL for non-seeded arteries demonstrates a weaker relationship (r2=0.39, P<0.001). The slope of 0.8±0.2 (95% CI 0.4 to 1.2) indicates a less uniform remodeling pattern.

 
3.6 SEM analysis
SEM was performed on paired arteries from two animals (day 21 and day 28). There was a striking difference in the appearance of the surface of seeded versus non-seeded vessels (Fig. 4). Seeded arteries demonstrated a confluent lining of endothelium over virtually all of the surface examined, with relatively sparse red cells and blood elements seen. In contrast, non-seeded arteries had large non-endothelialized areas with exposed smooth muscle. These areas were notable for the presence of numerous adherent mononuclear cells, platelets, and fibrinous debris.


Figure 4
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  		Fig. 4 Representative SEMs of (a) seeded and (b) non-seeded artery from rabbit sacrificed at 21 days after injury. The seeded vessel surface demonstrates a confluent, cobblestone endothelial pattern with occasional red blood cells. In contrast, the non-seeded artery surface exhibits large areas of exposed SMCs with adherent platelets, leukocytes, and fibrinous debris. (x480).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The overall goal of these experiments was to determine the effects of accelerating re-endothelialization (by EC seeding) on the structural remodeling of arteries subjected to severe mechanical and dietary injury. The results demonstrate improved angiographic luminal caliber and reduced overall wall thickening (intima+media) in EC-seeded vessels. EC-seeded vessels demonstrated greater uniformity in final lumen area and remodeling. These observations lend further support to the notion that restoration of the EC monolayer is an important biologic event in the pathophysiology of luminal narrowing following arterial injury.

In this rabbit model of balloon injury and diet-induced hypercholesterolemia, high grade stenoses were frequently observed in control vessels, often in juxtaposition to areas of luminal dilation. These stenoses were segmental, and were characterized predominantly by increased neointimal mass, though a remodeling effect was also apparent in that overall vessel size (EEL area) was an important modifier of final lumen area. These observations are in general agreement with those of other investigators who have employed arterial injury models in the atherosclerotic rabbit [1,3,14]. We speculate that the more uniform pattern of healing observed in seeded arteries may be related to a more uniform (temporal and spatial) pattern of re-endothelialization; this hypothesis requires further study.

Endothelial cell seeding has been extensively studied as a possible strategy for improving the long-term patency of small caliber prosthetic vascular grafts [15,16]. Although recent data suggests some potential benefit for patients requiring prosthetic infrainguinal bypass grafts [17,18], a clinical role for this approach remains unclear, particularly given its associated costs and technical complexity. Only a few studies have examined the utility of EC seeding in accelerating the healing of native vascular surfaces [19–21]. While the effects of EC seeding on the hyperplastic response of the vessel wall seem to be model-dependent, arterial surface properties including platelet deposition [21], barrier function [22], and adhesiveness for monocytes [22], have been beneficially altered experimentally.

The potential mechanism(s) by which restoration of the EC monolayer influences repair following arterial injury remains speculative and is the subject of ongoing study in our laboratory. The mechanical barrier function provided by a confluent endothelial monolayer may be critical for limiting deposition of proteins and lipid–protein complexes into the arterial wall where they may play a role in stimulating secondary inflammatory or thrombotic cascades. Alternatively, the endothelium may influence arterial healing by directly modulating the proliferation or phenotype of the underlying SMCs which form the bulk of the neointima. This notion is supported by a large body of experimental, predominantly in vitro data [23,24]. Another potential mechanism by which the endothelium may influence remodeling is through its tonic influence on the contractile state of the vascular smooth muscle, mediated by vasoactive substances such as nitric oxide and endothelin-1 [4,5].

The SEM images suggest that the recruitment of inflammatory cells and platelets to the healing arterial surface may be downregulated by the reconstituted endothelium. The potential role of the platelet and its products in restenosis has long been speculated [25,26]. Numerous lines of evidence suggest that the recruitment of inflammatory cells, particularly monocytes, to sites of vascular injury is a critical event in the pathogenesis of IH [27,28]. Monocytes may bind to the arterial wall through a number of mechanisms, including binding to deposited IgG in injured EC or non-endothelialized intima [29], as well as binding to specific cell surface adhesion molecules expressed by activated EC or SMC [30–33]. In a rabbit model of stent-induced arterial injury, heparin was shown to inhibit monocyte adhesion and infiltration; this effect was strongly correlated with its suppression of neointimal thickening [34]. A similar study of stented rabbit iliac arteries correlated the presence of ‘remnant’ endothelium at the stent site with reduced monocyte recruitment and decreased late intimal thickening [35]. One clinical study demonstrated a direct correlation between late lumen loss after coronary angioplasty and the activation status of circulating monocytes prior to the procedure [36]. In recent studies, we have demonstrated that EC seeding reduced the adhesion of THP-1 cells to injured rabbit arteries ex vivo [22]. The current investigations did not directly quantitate the influence of EC seeding on inflammatory cell recruitment in this model, which is the subject of ongoing kinetic studies.

In summary, our results suggest that venous endothelial cells, when implanted at sites of acute arterial injury, are capable of relining the flow surface and influencing lesion progression in the cholesterol-fed rabbit. Further experiments are necessary to examine the function of the neo-endothelium, the mechanisms of inflammatory cell recruitment during arterial healing, and the impact of endothelial restoration on the phenotype of the underlying SMCs. Approaches to accelerate native EC relining, using pro-angiogenic cytokines, merit ongoing study [37]. Alternatively, the EC seeding approach, while technically complex, may still be a potentially useful strategy, particularly if combined with other systemic or local therapies in a multi-tiered approach to control the arterial injury response.

Time for primary review 33 days.


   Acknowledgements
 
Supported in part by grants from the American Heart Association, Connecticut Affiliate (CT-96-GB-5) and the Ohse Foundation to MSC. We wish to acknowledge the technical support of Thomas Ardito of the Ultrastructure Facility at the Boyer Center for Molecular Medicine (scanning electron microscopy) and John Alderman of the Department of Radiology, Yale University School of Medicine (angiography). MSC is the recipient of a Mentored Clinical Scientist Development Award (1 K08 HL04189-01) from the National Heart, Lung and Blood Institute and the Lifeline Foundation.


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

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