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Cardiovascular Research 1997 36(3):396-407; doi:10.1016/S0008-6363(97)00168-5
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Predictors of luminal narrowing by neointima after angioplasty in atherosclerotic rabbits

S.David Gertza,*, William L Barryb, Lawrence W Gimpleb, Shmuel Banaia,c, Louise S Pereza, Coleen A McNamarab, Eric R Powersb, Michael Ragostab, Gary K Owensb, William C Robertsd and Ian J Sarembockb

aDepartment of Anatomy and Cell Biology, The Hebrew University-Hadassah Medical School, POB 12272, Jerusalem 91120, Israel
bThe Cardiovascular Division, University of Virginia, Charlottesville, VA, USA
cDepartment of Cardiology, Bikur Cholim Hospital, Jerusalem, Israel
dBaylor Cardiovascular Institute, Baylor University Medical Center, Dallas, TX, USA

* Corresponding author. Tel. (+972-2) 675-8446, -8422; Fax (+972-2) 675-7451, E-mail: gertz@yam-suff.cc.huji.ac.il

Received 9 January 1997; accepted 7 May 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The present study was designed to identify the predictors of cross-sectional area narrowing by neointima (%CSAN-N) after balloon angioplasty (BA) in the cholesterol fed rabbit model. Methods: Angiographic, histomorphometric, and immunohistochemical data were analyzed from 91 femoral arteries of New Zealand white rabbits. Focal atherosclerosis was induced by air desiccation of the endothelium followed by a 2% cholesterol diet for 28 days. The rabbits received heparin (150 U/kg) at the time of BA (2.5 mm; three, 60-second, 10-atm inflations). Arteries were perfusion-fixed and excised 7 (n = 16), 14 (n = 11), 21 (n = 9), or 28 (n = 20) days after BA. Non-angioplastied arteries were de-endothelialized (cholesterol-fed [n = 12] or normal diet [n = 8]), non-injured but cholesterol-fed (n = 7), or normal (n = 8). Results: Univariate regression across all groups showed that the absolute area of the lumen by histomorphometry (LA) correlated significantly with the area bounded by the external elastic lamina (EEL) (vessel size), but no correlation was found with the absolute area of neointima or media, the percentage disruption of the internal elastic lamina (IEL), or the percentage of neointima and media occupied by foam cells. However, %CSAN-N correlated significantly with the area bounded by the EEL, significantly with the absolute neointimal area, and negatively with the absolute LA (p<0.0001). Significant correlations were also found between %CSAN-N and the % IEL disrupted, the area of neointima and media occupied by RAM-11+ foam cells, and the loss of {alpha}-actin positivity in the media (p<0.0001). Conclusions: These studies show that neointimal formation contributes significantly to luminal narrowing 1 month after angioplasty in this model, that the degree of vascular injury and the extent of foam cell accumulation in the neointima and media are significant independent predictors of neointimal formation, and that the area of the neointima, and the percent narrowing by neointima, are important predictors of remodeling itself (EEL area). These predictors were not identifiable when the analysis was focused on the determinants of absolute luminal area alone.

KEYWORDS Angioplasty; Neointima; Restenosis; Remodeling; New Zealand white rabbits


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Discrepancies between success in experimental animals with a variety of pharmacologic strategies and failure with such agents in several clinical trials have raised questions concerning the mechanism of restenosis in humans [1, 2]. It has been suggested that the predominant mechanism responsible for the late reduction in luminal area after conventional balloon angioplasty in humans is remodeling of the arterial wall to produce a smaller overall vessel size [3, 4]rather than neointimal growth associated with smooth muscle cell proliferation [5]. Some reports in experimental animals and humans have concluded that the degree of arterial enlargement is more important than neointimal area in determining late lumen size [6], and that factors related to arterial remodeling rather than neointimal growth may dominate the response to angioplasty [4, 7]. Other reports have shown that restenosis after angioplasty results from both intimal hyperplasia and arterial remodeling [8–10], and that it is the balance between the two, which results in either constriction or "compensatory" enlargement, that determines the final luminal size after angioplasty [11, 12]. Studies from our group using the double injury cholesterol-fed rabbit model have suggested that although remodeling to a larger or smaller arterial size after angioplasty does occur, it does not appear to account for the increase in cross-sectional area narrowing by neointima, or the reduction in absolute luminal area, in most of the individual arteries in this model [13–15].

Effective antithrombotic/antiplatelet therapy has profoundly decreased the frequency of thrombosis, and properly deployed stents have nearly eliminated the occurrence of recoil [16]. The overall rates of restenosis following stent deployment have improved to below 20–30% of those treated [17–21], but late luminal loss has still been shown to be due predominantly to intimal hyperplasia [22, 23]. This underscored the need for continued effort to identify factors responsible for this proliferative/synthetic process. The current study was therefore designed to identify by correlative angiographic, histomorphometric, and immunohistochemical analyses, the predictive correlates of cross-sectional area narrowing by neointima after balloon angioplasty (BA) in the cholesterol fed rabbit model.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Study design
Forty seven New Zealand White rabbits were anesthetized by intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg). Rabbits were assigned at random to one of four angioplasty groups and five baseline (non-angioplasty) groups (Fig. 1). Of the 94 femoral arteries, 3 were not useable due to surgical (2 arteries) or preparative (1 artery) accidents leaving 91 arteries for these studies. Atherosclerosis was induced in a 1 to 2 cm segment of the femoral arteries assigned to be angioplastied by a combination of nitrogen air desiccation of the endothelium and a 2% cholesterol, 6% peanut oil diet for 1 month [24–26]. Animals undergoing angioplasty received single bolus heparin (150 U/kg heparin sodium injection, porcine intestinal mucosa, 1000 USP U/ml; Solopak Laboratories, Franklin Park, IL). The angioplastied femoral arteries were perfusion fixed and excised 7 days (n = 16), 14 days (n = 11), 21 days (n = 9) or 28 days (n = 20) after the procedure. Baseline groups did not undergo angioplasty but received bolus heparin at the time of endothelial injury. The non-angioplastied groups were focally de-endothelialized, fed cholesterol for 28 days, and sacrificed at the end of the feeding period (n = 7) or 28 days later (total of 56 days from time of endothelial injury) (n = 5); fed cholesterol but not de-endothelialized and sacrificed at the end of the 28-day feeding period (n = 7); de-endothelialized but not fed cholesterol and sacrificed after 28 days (n = 8); or normal animals which were not injured or fed and were sacrificed on arrival (n = 8).


Figure 1
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  		Fig. 1 Study design.

 
2.2 Balloon angioplasty
Twenty eight days after endothelial injury and cholesterol feeding, a 5 F Berman angiographic balloon catheter (Arrow International, Inc., Reading, PA) was inserted into the right common carotid artery and advanced to two vertebral segments proximal to the aortoiliac bifurcation. After administration of heparin, baseline angiograms of the femoral arteries were obtained as reported previously [25, 27]. Balloon angioplasty was performed under fluoroscopic guidance 28 days after endothelial injury and cholesterol feeding using a 2.5 mm balloon dilation catheter (Advanced Cardiovascular systems, Inc.). Three, 60-second, 10-atm inflations were performed at 60-second intervals using a hand inflator as described previously [24, 25]. The in vivo balloon dimension was recorded. The mean ratio of balloon diameter to the average of normal arterial diameters proximal and distal to the stenosis was 1.3±0.2. This slight "oversizing" provided for the deep arterial injury as reported in detail previously [24, 26]. After the post-procedural angiogram, the catheter system was removed, the carotid arteries ligated, and the wound sutured. Final angiograms were performed immediately prior to sacrifice by way of the contralateral carotid artery.

2.3 Analysis of angiograms
Quantitative angiographic measurements were performed by blinded analysis using a computer-assisted system described previously [26, 27]. Minimum luminal diameters (MLD) were measured at the site of stenosis and at segments proximal and distal to the stenosis. The site measured for restenosis was that which showed the maximal diameter reduction on the pre-angioplasty angiogram even if this was not the most severely narrowed point on subsequent angiograms. Angiographic data on luminal diameter reduction (LDR) were computed relative to proximal and distal reference points. Percent LDR=[MLD at sacrifice x100÷average of proximal and distal reference points] –100.

2.4 Preparation of specimens for histomorphometry
Following the final angiogram, and with the angiographic catheter positioned above the aortoiliac bifurcation, the animals were administered an overdose of Nembutal (sodium pentobarbital). The distal arterial tree was then pressure perfused with 4% buffered formaldehyde (100 ml, 100 mmHg, room temperature, for 15 minutes). Segments of the femoral arteries (4 to 5 cm in length) were excised bilaterally, postfixed in 4% formaldehyde, cut in cross section at 1 to 2 mm intervals, dehydrated in increasing concentrations of ethanol and xylene, and embedded in paraffin.

Sections (5 µm) were stained with hematoxylin–eosin and by the Movat pentachrome method [28]. Adjacent sections were stained immunohistochemically with RAM-11 (mouse anti-rabbit monocyte/macrophage) and antibodies to smooth muscle cell {alpha}-actin (mouse anti-human) as follows: Sections were deparaffinized at 60°C for 10 minutes, hydrated in deionized water, and digested in protease VIII solution in phosphate buffer (RAM-11 only) (37°C) (Sigma Cat # P5380). Sections were then blocked with 3% hydrogen peroxide for 10 minutes and washed in phosphate buffered saline (PBS) (2 changes, 5 minutes each). After blocking with horse serum for 20 minutes (Vector Laboratories), the primary antibody, RAM-11 (Dako, Cat # M633) (1:10 dilution) or {alpha}-actin (ENZD Diagnostics, Cat. # C 34931) (1:200), was applied for 1 hour. The secondary antibody was a biotinylated horse anti-mouse antibody (30 minutes) (Vector Laboratories, Cat # PK-4002). This was followed by PBS wash, "ABC" reagent (30 minutes) (Vector Laboratories, Cat # PK-4000), PBS wash, and DAB substrate (4 minutes) (Sigma Fast DAB tablets, Cat # D4418). Sections were counterstained with Harris hematoxylin (1 minute).

2.5 Quantitative histopathology
Histopathological analysis was performed by observers blinded to treatment group. The section with the greatest luminal narrowing by neointima was identified from each femoral arterial segment. For each section, the absolute histomorphometric measurements included: luminal area (LA), neointimal area, medial area, area bounded by the internal elastic lamina (IEL), area bounded by the external elastic lamina (EEL) (overall vessel size), and components of the neointima and media. Derived calculations and observations included the following:

Percent Cross-Sectional-Area-Narrowing by Neointima (%CSAN-N)=[area bounded by the IEL–luminal area]x100÷area bounded by the IEL

Percent Cross-Sectional-Area-Narrowing by Neointima Plus Media (%CSAN-N+M)=[area bounded by the EEL–luminal area]x100÷area bounded by the EEL

Intima to Media Area Ratio (I/M ratio)=absolute area of the neointima÷absolute area of the media.

Percentage of total vessel area occupied by media=absolute medial areax100÷area bounded by EEL.

2.6 Arterial injury
Sections were characterized with respect to histologic evidence of plaque tear (up to the IEL, into the media, into the adventitia, or complete perforation) and presence of luminal thrombus. The extent of disruption of the IEL was determined by linear measurement of the defect and calculation of its percentage of the total length of the IEL.

2.7 Components of neointima and media
The overall percentage of neointima occupied by foam cells was calculated by computerized planimetry of RAM-11-stained sections.

The distribution of RAM-11 positive foam cells within the intima was assessed by dividing the intima into inner (luminal), middle, and outer thirds (intima-A, B, or C; see Table 3) and estimating what percentage of all intimal foam cells is found in each of the three areas (0–25%, 26–50%, 51–75%, or 76–100%). An intimal foam cell score of 1, 2, 3, or 4 was assigned respectively for semiquantitative evaluation.


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  		Table 3 Distribution of foam cells and smooth muscle cells within the intima after angioplasty

 
The extent of foam cell "accumulation" in the media was assessed by subdividing the media into four equal quadrants and estimating whether RAM-11 positive foam cells occupied 0–25%, 26–50%, 51–75%, or 76–100% of the media. A score of 1, 2, 3, or 4 was assigned respectively for this semiquantitative analysis. This method of assessment of medial foam cells was chosen rather than direct planimetric tracing, since the medial foam cells were not distributed in discrete groups like those found in the intima [26].

The distribution of {alpha}-actin positive smooth muscle cells in the neointima and media were assessed qualitatively and semi-quantitatively in the same manner as the RAM-11 positive foam cells.

Histomorphometric measurements were performed using a CUE-2 Image Analyzer (Galai Production, Ltd., Tel-Aviv, Israel) in association with an Olympus BH-2 microprojection system as described previously [27].

2.8 Statistical analysis
Data are reported as the number of femoral arteries in each treatment group and expressed as mean±standard deviation. Absolute areas are presented as µm2 x10–4. The effect of survival time on individual angiographic and histopathologic parameters was assessed by one-way ANOVA followed by the Fisher Protected Least Significant Difference test for multiple comparisons as the post hoc test with 95% and 99% multicomparison significance levels. Distribution of RAM-11 positive foam cells and {alpha}-actin positive smooth muscle cells by intimal zone (A, B or C) over the 4 survival times was assessed by two-factor, repeated measurements ANOVA. Comparison of categorical data was made by coded {chi}2 contingency analysis. Positive and negative correlates of cross-sectional area narrowing by neointima and other histomorphometric parameters were determined from univariate regression analyses. Probabilities were calculated from ANOVA. Since it cannot be determined, a priori, that the various histomorphometric variables are related or unrelated, multiple regression analysis was not used. The Statview II statistical package (Brain Power, Inc.) was used for these calculations.

This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85–23, revised 1985).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
The principal component of the neointima in all angioplastied and de-endothelialized, non-angioplastied arteries is fibromuscular tissue (smooth muscle cells with abundant extracellular matrix) which occupies approximately 90% of the neointimal area (Fig. 2). Non-injured arteries had no neointima despite cholesterol feeding.


Figure 2
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  		Fig. 2 Femoral artery of a rabbit subjected to endothelial injury, cholesterol feeding for 28 days, balloon angioplasty, and sacrifice 28 days later showing the intima consisting primarily of fibromuscular tissue with foam cells concentrated primarily in the deeper portion of the intima and media. L=lumen, M=media, IEL=internal elastic lamina, FC=foam cells. Movat pentachrome stain, x80.

 
3.1 Arterial injury
Histologic evidence of plaque tear was found in 49 (88%) of the 56 angioplastied arteries. Of these, 47 (96%) had histologic evidence of damage reaching the media, with disruption the IEL, and 17 of these (30% of all arteries) had evidence of damage reaching the adventitia, with disruption of the EEL. Disruption of the IEL was found in angioplastied as well as non-angioplastied arteries of animals which were de-endothelialized and fed cholesterol for 28 days and sacrificed 28 days later (Fig. 3). However, the percent of the IEL missing was significantly less in non-angioplastied arteries (BA, 40±24% vs. no-BA, 5±8%, p<0.01 by 1-way ANOVA with Fisher PLSD as post hoc) (Table 1Table 2). Of the 56 angioplastied arteries, only 4 (7%) had luminal thrombi, and all were found in arteries excised 7 days after angioplasty.


Figure 3
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  		Fig. 3 Femoral artery of a cholesterol fed rabbit 28 days after angioplasty (double injury) showing contiguity of foam cells (intermingled with fibrous tissue and smooth muscle cells) between the media and intima through the defect in the IEL. IEL=internal elastic lamina, M=Media. Movat stain, x240.

 

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  		Table 1 Angioplastied arteries

 

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  		Table 2 Non-angioplastied arteries

 
3.2 Absolute luminal area vs. cross-sectional area narrowing by neointima
By angiography, the absolute mean minimum luminal diameter at the site of angioplasty at the time of sacrifice did not change significantly from 7 to 28 days after the procedure (p = 0.38 by 1-way ANOVA over the 4 time periods) (Table 1). However, the mean percent luminal diameter reduction at the time of sacrifice among angioplastied animals increased significantly over time after the procedure (p = 0.01) (Table 1). Likewise, by histomorphometry, the mean absolute area of the lumen at the site of angioplasty (LA) did not change significantly from 7 to 28 days after angioplasty (p = 0.40). However, significant increases were found over time in the percent by which the cross-sectional area of the lumen is narrowed by neointima (%CSAN-N) (p = 0.009), the intima to media area ratio (I/M ratio) (p = 0.0002), and the number of arteries narrowed greater than 50% in cross-sectional area by neointima (n>50% CSAN-N) (p = 0.01 by total {chi}2 over all groups) (Table 1).

Univariate regression analysis over all groups (angioplastied and non-angioplastied) showed a significant positive correlation between absolute LA and area bounded by the IEL (r = 0.54, df=90, p = 0.0001) and area bounded by the EEL (overall vessel size) (r = 0.45, df=90, p = 0.0001), but no significant correlation was found between LA and absolute neointimal area (r = 0.025, df=90, p = 0.81) (Fig. 4). In addition, no significant correlations were found between the absolute LA and the absolute medial area (r = 0.023, df=90, p = 0.83), the percent disruption of the IEL (r = 0.013, df=90, p = 0.90), the percent of neointimal area occupied by RAM-11+ foam cells (r = 0.08, df=77, p = 0.48), the RAM-11+ medial foam cell score (see Section 2) (r = 0.21, df=89, p = 0.05), or {alpha}-actin positivity of media (see Section 2) (r = 0.06, df=88, p = 0.57). On the other hand, when focusing on %CSAN-N, we find a significant positive correlation not only with vessel size (area bounded by the EEL) (r = 0.51, df=90, p = 0.0001), but also with the absolute area of the neointima (r = 0.75, df=90, p = 0.0001) (Fig. 3). Moreover, a significant negative correlation was found with absolute LA over all groups (r = 0.41, df=90, p = 0.0001), or when the analysis was restricted to just the 4 angioplasty groups (r = 0.59, df=55, p = 0.0001) (Fig. 5), indicating that the greater the %CSAN-N, the smaller the area of residual lumen.


Figure 4
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  		Fig. 4 Scattergrams with regression lines showing: top, left, significant positive correlation, over all groups, between the absolute luminal area and the area bounded by the external elastic lamina (EEL AREA) (overall vessel size) (r = 0.45, df=90, p = 0.0001), but, bottom, left, no significant correlation was found between luminal area and absolute neointimal area (NEO AREA) (r = 0.025, df=90, p = 0.81). On the other hand, significant positive correlations were found, top, right, between percent cross-sectional area narrowing by neointima (%CSAN-N) and EEL AREA (r = 0.51, df=90, p = 0.0001), and, bottom, right, between %CSAN-N and the absolute neointimal area (r = 0.75, df=90, p = 0.0001). Area units=10–4 µm2.

 

Figure 5
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  		Fig. 5 Scattergram with regression line showing a significant negative correlation over the four angioplastied groups between percent cross-sectional area narrowing by neointima (%CSAN-N) and absolute luminal area (r = 0.59, df=55, p = 0.0001). Area units=10–4 µm2.

 
The %CSAN-N also correlated significantly (in ascending order) with the % LDR at sacrifice by angiography (r = 0.40, df=78, p = 0.0004), the % of IEL which was disrupted (r = 0.53, df=90, p = 0.0001), the RAM-11+ medial foam cell score (r = 0.55, df=89, p = 0.0001), the area of neointima occupied by RAM-11+ foam cells (r = 0.50, df=77, p = 0.0001), the absolute area of the media (r = 0.6, df=90, p = 0.0001), and the loss of {alpha}-actin positivity of media (r = 0.65, df=88, p = 0.0001).

Arteries excised 28 days after angioplasty were significantly more narrowed by neointima than non-angioplastied control arteries from animals which received the same atherogenic regimen and were sacrificed at the same time period (%CSAN-N: BA, 60±21 vs. no-BA, 14±14, p<0.01; I/M ratio: BA, 2.4±1.2 vs. no-BA, 0.60±0.74, p<0.001) (by 1-way ANOVA with Fisher PLSD) (Tables 1 and 2Go). The percent narrowing by neointima in non-angioplastied control arteries excised 28 days after termination of cholesterol feeding was not significantly different from non-angioplastied arteries excised immediately after termination of the atherogenic regimen (without waiting the additional 28 days) (Table 2). The %CSAN-N of non-angioplastied, de-endothelialized arteries from animals fed cholesterol for 28 days was similar to that of non-angioplastied, de-endothelialized arteries from animals not fed cholesterol, but significantly greater than those which were fed cholesterol for 28 days without initial endothelial injury (Table 2). This is in agreement with the concept that cholesterol feeding alone (without endothelial injury) is insufficient to produce atherosclerotic lesions with luminal narrowing significantly different from normal arteries in this model.

3.3 Medial area
Among angioplastied arteries, the medial area appears to decrease over time resulting in its being a larger percentage of the total area bounded by the EEL at 7 days than at 28 days (Table 1). Univariate regression analysis over all groups (angioplastied and non-angioplastied) showed significant positive correlations between the absolute area of the media and absolute area of the neointima (r = 0.54, df=90, p = 0.0001) and %CSAN-N (r = 0.56, df=90, p = 0.0001) but not with the absolute luminal area (r = 0.023, df=90, p = 0.83). Significant positive correlations were also noted across all groups between absolute medial area and %RAM-11+ FC-N (r = 0.55, df=77, p = 0.0001), the RAM-11+ medial foam cell score (r = 0.55, df=89, p = 0.0001), and overall vessel size (EEL area) (r = 0.66, df=90, p = 0.0001). A significant negative correlation was found with the {alpha}-actin positivity score of the media (r = 0.60, df=88, p = 0.0001).

3.4 Arterial size
A significant positive correlation was found between arterial size and IEL area (r = 0.97, df=90, p = 0.0001), absolute LA (see above), absolute area of the neointima (r = 0.87, df=90, p = 0.0001), absolute medial area (see above), %CSAN-N (see above), percent of the IEL which was missing (r = 0.43, df=90, p = 0.0001), %RAM-11+ FC-N (r = 0.45, df=77, p = 0.0001), and RAM-11+ medial foam cell score (r = 0.32, df=89, p = 0.32). A negative correlation was found between arterial size and the {alpha}-actin positivity of the media (r = 0.59, df=88, p = 0.0001).

3.5 Distribution of foam cells in the intima
The mean percents of neointimal area occupied by foam cells in the various groups are detailed in Tables 1 and 2Go. The intimal RAM-11+ foam cell score (see Section 2) was higher in the outer (abluminal) portions of the neointima (i.e. closer to the IEL than the lumen) (Table 3, Fig. 6). Among angioplastied arteries, no significant differences were found in the absolute area or in the percentages of the total neointimal area occupied by RAM-11 positive foam cells (%RAM-11+FC-N) over the four survival periods (Table 1). The %RAM-11+FC-N of non-angioplastied arteries which had endothelial injury plus cholesterol feeding for 28 days, but were excised 28 days later, was also not different from similar non-angioplastied, de-endothelialized arteries excised immediately after cholesterol feeding (Table 2). No foam cells were found in the intima of normal arteries, very few foam cells were found in rabbits which had endothelial injury but were not fed cholesterol, and foam cells were not found in the intima of animals fed cholesterol without endothelial injury.


Figure 6
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  		Fig. 6 Femoral artery of a cholesterol fed rabbit 28 days after angioplasty (double injury). Left, {alpha}-actin stain showing distribution of the {alpha}-actin positive smooth muscle cells in the intima and media. x240. Right, RAM-11 stain showing extensive foam cell accumulation in the media and adjacent intima. x210. L=lumen, arrow=internal elastic lamina.

 
Univariate regression analysis across all treatment groups showed no correlation between the percent of total neointima area occupied by foam cells and absolute LA (r = 0.08, df=77, p = 0.48), but a significant correlation with %CSAN-N (r = 0.50, df=77, p = 0.0001).

3.6 Foam cells in the media
A striking feature of the rabbit angioplasty model is the abundant infiltration of foam cells into the media which ranged from approximately 25% to a situation where the majority of the media often appeared to be replaced by foam cells. The medial foam cells were intermingled with fibrous tissue and smooth muscle cells and contiguous through the defect in the internal elastic lamina with the foam cells of the intima (Figs. 3 and 6Go). Foam cells were not found in the media of vessels of animals fed cholesterol without prior endothelial injury or subsequent angioplasty.

The percent of media occupied by RAM-11+ foam cells in angioplastied arteries increased significantly from 7 to 21 days after the procedure (Table 1). No foam cells were found in the media of non-angioplastied, non de-endothelialized arteries despite cholesterol feeding, or in those which has endothelial injury without cholesterol feeding (Table 2).

Regression analysis showed that the RAM-11+ medial foam cell score correlated poorly with the absolute LA (r = 0.21, df=89, p = 0.05) but significantly with %CSAN-N (r = 0.55, df=89, p = 0.0001). A significant positive correlation was also found with the percent which the media occupies of the total EEL area (r = 0.38, df=88, p = 0.0002), and the percent of RAM-11+ foam cells in the neointima (r = 0.4, df=85, p = 0.0001).

3.7 Smooth muscle cells
The overall distribution of {alpha}-actin-positive smooth muscle cells in the neointima of angioplastied arteries appeared more homogeneous than that of the foam cells; but, as expected, a slight "preference" was noticed for luminal rather than abluminal areas (Fig. 6). The {alpha}-actin-positivity score of angioplastied arteries appeared to decrease over the four time periods with a significant difference between 7 and 28 days (Table 1) (p = 0.04 over all groups by 1-way ANOVA). Among animals which had endothelial injury, cholesterol feeding for 28 days and were sacrificed 28 days later, the medial {alpha}-actin positivity score of those undergoing balloon angioplasty was significantly lower than those without angioplasty (1.6±.9 vs. 2.8±1.6, p<0.05 by 1-way ANOVA with Fisher PLSD) (Tables 1 and 2Go).

No significant correlation was found between the {alpha}-actin positivity score of the media and absolute LA (r = 0.06, df=88, p = 0.57), but a significant negative correlation was found with %CSAN-N (r = 0.65, df=88, p = 0.0001). Significant negative correlations were also found with the percent IEL missing (r = 0.5, df=88, p = 0.0001), absolute area of neointima (r = 0.62, df=88, p = 0.0001), the RAM-11+ medial foam cell score (r = 0.6, df=90, p = 0.0001), and the overall vessel size (EEL area) (r = 0.6, df=88, p = 0.0001).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We have shown that the absolute area of the lumen correlated significantly with the overall vessel size. However, no significant correlation was found between absolute luminal area and absolute neointimal area. On the other hand, the percent cross-sectional area narrowing by neointima correlated positively with the vessel wall size, and the absolute area of the neointima, but correlated negatively with the absolute LA. Moreover, LA, absolute neointimal area, IEL disruption, area of the neointima occupied by RAM-11+foam cells, area of the media occupied by foam cells, and the loss of {alpha}-actin positivity of the media appeared to be strong, independent predictors of %CSAN-N (p<0.0001). These predictors were not identifiable when the analysis was focused on the determinants of absolute luminal area alone.

In an earlier study from our group using the double injury cholesterol-fed rabbit femoral artery model with different interventional strategies including balloon only, laser plus balloon, no balloon and balloon with administration of the factor Xa inhibitor antistasin [13], we showed that although remodeling to a larger or smaller arterial size after angioplasty does occur, it does not appear to account for the reduction in absolute luminal area at the site of the procedure in most cases when the arteries are analyzed individually. We found that remodeling to a smaller overall vessel size was present in only 8 (16%) of 51 individual arteries at the site of restenosis when each is compared to its respective adjacent reference segment. The overall arterial size was larger in 12 (24%) of 51 cases and not significantly different from its adjacent segment in 31 (61%) of 51 cases. On the other hand the %CSAN-N 28 days after angioplasty was significantly greater than the neointimal narrowing of adjacent nonangioplastied segments of the same arteries, greater than de-endothelialized, nonangioplastied control arteries of cholesterol fed animals, and greater than angioplastied segments (de-endothelialized plus cholesterol feeding) of animals treated with the factor Xa inhibitor antistasin. From that study we learned that although remodeling of the arterial wall to a smaller or larger vessel size can occur, it does not appear to account for the increase in cross-sectional area narrowing by neointima, or the reduction in absolute luminal area, in the great majority of vessels after one month in this model when rigorous intra-arterial and inter-group controls are used.

In a recent study of the response of 17 normal porcine coronary arteries to severe balloon inflation and withdrawal, Andersen et al. [4]also reported that although absolute lumen size correlated significantly with overall vessel size, it did not correlate with absolute neointimal area. They and others performing similar analyses concluded therefore that neointimal formation did not explain luminal narrowing after deep vessel wall injury and emphasized the importance of remodeling (change in overall EEL area). However, it is precisely the variations in overall vessel size which appear to have contributed to this poor correlation in these and in the current study. The current study focused on the percent which the neointima occupies of the total area bounded by the IEL (%CSAN-N) which essentially normalizes the effect of overall arterial wall size. In so doing, we found that the %CSAN-N increased with vessel size and with absolute neointimal area, but correlated negatively with absolute LA, demonstrating that neointimal formation, in fact, contributes substantially to luminal narrowing in this rabbit model.

Furthermore, although remodeling to a larger vessel size has been shown to occur in the current study and previous studies from our group, we find that the absolute LA (r = 0.87, df=90, p<0.0001) and %CSAN-N (r = 0.51, df=90, p<0.0001) are actually important predictors of the remodeling itself (overall EEL area) in this model reemphasizing the importance of identifying factors responsible for neointimal growth after angioplasty.

In the current study, we found that the extent of luminal narrowing by neointima correlated significantly with the percentage of the IEL which was disrupted, emphasizing that the more severe the injury to the wall, the greater the neointimal growth. Among animals which had endothelial injury and cholesterol feeding, the percent IEL missing was significantly greater in angioplastied than non-angioplastied arteries. Thus, although disruption of the IEL occurs during atherogenesis in this model, possibly in part from mechanical damage at the time of the initial endothelial cell injury, considerable additional damage is done to the IEL as a result of the balloon angioplasty procedure.

A significant positive correlation was also found between the %CSAN-N and the area of neointima and media occupied by RAM-11+foam cells. The distribution of the foam cells in this model was such that a significantly greater concentration of cells was found in the outer (abluminal) portion of the neointima, closer to the IEL. We have seen here, and in previous studies using this double injury model, that the foam cells of the intima are contiguous with foam cells of the media and adventitia, and can often be found in association with neovascular channels passing between the adventitia and media [26]. This distribution is in agreement with that reported recently by others [10], and it raises some questions concerning the conventional thinking of an exclusive luminal origin of these cells [29]. It appears from our studies and those of Wilensky [10]that the additional injury applied by angioplasty facilitates additional migration of foam cells into the media, possibly from the intima and adventitia. This may add to the proliferative/synthetic process by release of a variety of vasoactive cytokines from these cells which may promote migration and/or proliferation of arterial smooth muscle cells [30]. These monocyte–macrophage type foam cells have also been shown to be capable of releasing a variety of proteolytic enzymes and metaloproteinases which may also facilitate neointimal growth by degradation of extracellular matrix proteins permitting subsequent restructuring of this matrix or "remodeling" [31–33].

We found that the %CSAN-N correlated strongly with the reduction in {alpha}-actin positivity of the media. This loss of {alpha}-actin positivity may be a reflection of increased necrosis or apoptosis [34]of the medial smooth muscle cells after the procedure with subsequent "accumulation" of RAM-11-positive foam cells, the great majority of which are {alpha}-actin negative, or due, at least in part, to the presence of other cell types which do not elaborate contractile proteins [35]. The absence of {alpha}-actin positivity has also been seen by us and others in cells which appeared to be smooth muscle cells with apparently normal nuclear and cytoplasmic morphology. This may be due to an actual reduction in {alpha}-actin antigenicity of the smooth muscle cells themselves which has been suggested to occur during accelerated elaboration by these cells of matrix proteins, chemoattractants, and expression of adhesion molecules [33, 36]. This would also be in accordance with the concept that smooth muscle cells express alternate isoforms of actin during contractile ({alpha}-isoform) and synthetic (β and {gamma}-isoforms) phases [37].

In conclusion, these studies show that neointimal formation contributes significantly to luminal narrowing one month after angioplasty in this model, that the degree of vascular injury and the extent of foam cell accumulation in the neointima and media are significant independent predictors of neointimal formation, and that the area of the neointima, and the percent narrowing by neointima, are important predictors of remodeling itself (EEL area). These predictors were not identifiable when the analysis was focused on the determinants of absolute luminal area alone. We cannot, and it was not our intention to, exclude the view that changes in vessel size might contribute to changes in luminal area after more prolonged survival times following angioplasty. Nonetheless, these studies emphasize that the approach "remodeling or neointimal formation" has a major danger of neglecting relevant and potentially treatable cellular processes which may be common to both mechanisms. We must be careful to prevent letting the pendulum swing away from efforts to prevent intimal hyperplasia. This is particularly true in this era of extensive use of intravascular stents, where geometric remodeling to a smaller arterial size is virtually eliminated, and where restenosis in properly deployed stents has been shown to be due predominantly to the formation of neointima.

Time for primary review 27 days.


   Acknowledgements
 
This work was supported in part by the Department of Health and Human Services, Research Grant (NIH) (RO1-HL-7849) (IJS), the Joint Fund of the Israel Ministry of Science and Arts and The German Ministry of Science, Technology, and Education (BMBF) DISMED87/1338GR (SDG, SB), American Heart Association, Virginia Affiliate (LWG). Dr. Gertz holds the Lillian and Rebecca Chutick Chair of Cardiac Studies, The Hebrew University, Jerusalem.


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

  1. Glagov S. Intimal hyperplasia, vascular modeling, and the restenosis problem. Circulation (1994) 89:2888–2891.[Free Full Text]
  2. Isner J.M. Vascular remodeling. Honey, I think I shrunk the artery. [editorial]. Circulation (1994) 89:2937–2941.[Free Full Text]
  3. Mintz G.S., Pichard A.D., Kent K.M., Satler L.F., Popma J.J., Leon M.B. Intravascular ultrasound comparison of restenotic and de novo coronary artery narrowings. Am J Cardiol (1994) 74:1278–1280.[CrossRef][Web of Science][Medline]
  4. Andersen H.R., Maeng M., Thorwest M., Falk E. Remodeling rather than neointimal formation explains luminal narrowing after deep vessel wall injury. Insights from a porcine coronary (Re)stenosis model. Circulation (1996) 93:1717–1724.
  5. Ragosta M., Barry W., Gimple L.W., Gertz S.D., McCoy K.W., Stouffer G.A., McNamara C.A., Powers E.R., Owens G.K., Sarembock I.J. Effect of thrombin inhibition with desulfatohirudin on the early kinetics of cellular proliferation after balloon angioplasty in atherosclerotic rabbits. Circulation (1996) 93:1194–1200.[Abstract/Free Full Text]
  6. Kakuta T., Currier J.W., Haudenschild C., Ryan T., Faxon D.P. Differences in compensatory vessel enlargement, not intimal formation, account for restenosis after angioplasty in the hypercholesterolemic rabbit model. Circulation (1994) 89:2809–2815.[Abstract/Free Full Text]
  7. Lafont A., Guzman L.A., Whitlow P.L., Goormastic M., Cornhill J.F., Chisolm G.M. Restenosis after experimental angioplasty. intimal, medial, and adventitial changes associated with constrictive remodeling. Circ Res (1995) 76:996–1002.[Abstract/Free Full Text]
  8. Post M.J., Borst C., Kuntz R.E. The relative importance of arterial remodeling compared with intimal hyperplasia in lumen renarrowing after balloon angioplasty. A study in the normal rabbit and the hypercholesterolemic Yucatan micropig. Circulation (1994) 89:2816–2821.[Abstract/Free Full Text]
  9. Mintz G.S., Popma J.J., Pichard A.D., Kent K.M., Satler L.F., Chien Chuang Y.A., Griffin J., Leon M.B. Intravascular ultrasound predictors of restenosis after percutaneous transcatheter coronary revascularization. J Am Coll Cardiol (1996) 27:1678–1687.[Abstract]
  10. Wilensky R.L., March K.L., Gradus-Pizlo I., Sandusky G., Fineberg N., Hathaway D.R. Vascular injury, repair, and restenosis after percutaneous transluminal angioplasty in the atherosclerotic rabbit. Circulation (1995) 92:2995–3005.[Abstract/Free Full Text]
  11. Currier J.W., Faxon D.P., Lafont A. Geometric remodeling and restenosis. Circulation (1995) 92:3581.[Web of Science][Medline]
  12. Nishioka T., Luo H., Eigler N.L., Berglund H., Kim C.-J., Siegel R.J. Contribution of inadequate compensatory enlargement to development of human coronary artery stenosis: an in vivo intravascular ultrasound study. J Am Coll Cardiol (1996) 27:1571–1576.[Abstract]
  13. Gertz S.D., Gimple L.W., Banai S., Ragosta M., Powers E.R., Roberts W.C., Perez L.S., Sarembock I.J. Geometric remodeling is not the principal pathogenetic process in restenosis after balloon angioplasty. Evidence from correlative angiographic-histomorphometric studies of atherosclerotic arteries in rabbits. Circulation (1994) 90:3001–3008.[Abstract/Free Full Text]
  14. Gertz S.D., Gimple L.W., Banai S., Ragosta M., Powers E.R., Roberts W.C., Perez L.S., Sarembock I.J. Geometric remodeling and restenosis. Reply to letter by Post et al. Circulation (1995) 92:2002–2003.[Web of Science][Medline]
  15. Gertz S.D., Gimple L.W., Ragosta M., Powers E.R., Sarembock I.J., Banai S., Perez L.S., Roberts W.C. Geometric remodeling. Reply to letter by Currier et al. Circulation (1995) 92:3581–3583.[Web of Science][Medline]
  16. Fuster V., Falk E., Fallon J.T., Badimon L., Chesebro J.H., Badimon J.J. The three processes leading to post PTCA restenosis: Dependence on the lesion substrate. Thrombos Haemostas (1995) 74:552–559.[Web of Science][Medline]
  17. Serruys P.W., de-Jaegere P., Kiemeneij F., Macaya C., Rutsch W., Heyndrickx G., Emanuelsson H., Marco J., Legrand V., Materne P., Belardi J., Stigwart U., Colombo A., Goy J.J., Van Den Heuvel P., Delcan J., Morer M.-A. A comparison of balloon-expandable-stent implantation with balloon angioplasty in patients with coronary artery disease. Benestent Study Group. N Engl J Med (1994) 331:489–495.[Abstract/Free Full Text]
  18. Serruys, W. Benestent-II pilot study: 6 months follow-up of phase 1,2, and 3. Circulation. 1995;92(Suppl):I-542-543. (abstract).
  19. Savage M.P., Fischman D.L., Schatz R.A., Teirstein P.S., Leon M.B., Baim D., Ellis S.G., Topol E.J., Hirshfeld J.W., Cleman M.W., Buchbinder M., Bailey S., Heuser R., Walker C.M., Curry R.C., Gebhardt S., Rake R., Goldberg S. Long-term angiographic and clinical outcome after implantation of a balloon-expandable stent in the native coronary circulation. Palmaz-Schatz Stent Study Group. J Am Coll Cardiol (1994) 24:1207–1212.[Abstract]
  20. Azar AJ, Detre K, Goldberg S, Kiemeneij F, Leon MB, Serruys PW. A Meta-Analysis on the clinical and angiographic outcomes of stents vs. PTCA in the different coronary vessel sizes in the Benestent-1 and Stress-1/2 trials. Circulation. 1995;92(Suppl):I-475. (abstract).
  21. Akira I, Hall P, Maiello L, Blengino S, Finci L, Ferraro M, Martini G, Colombo A. Coronary stenting of long lesions (greater than 20 mm) — a matched comparison of different stents. Circulation. 1995;92:I-688, 1995 (abstract).
  22. Gordon P.C., Gibson C.M., Cohen D.J., Carrozza J.P., Kuntz R.E., Baim D.S. Mechanisms of restenosis and redilation within coronary stents — quantitative angiographic assessment. J Am Coll Cardiol (1993) 21:1166–1174.[Abstract]
  23. Hoffmann R., Mintz G.S., Dussaillant G.R., Popma J.J., Pichard A.D., Satler L.F., Kent K.M., Griffin J., Leon M.B. Patterns and mechanisms of in-stent restenosis: A serial intravascular ultrasound study. Circulation (1996) 94:1247–1254.[Abstract/Free Full Text]
  24. Sarembock I.J., LaVeau P.J., Sigal S.L., Timms I., Sussman J., Haudenschild C., Ezekowitz M.D. The influence of inflation pressure and balloon size on the development of intimal hyperplasia following balloon angioplasty: A study in the atherosclerotic rabbit. Circulation (1989) 80:1029–1040.[Abstract/Free Full Text]
  25. Sarembock I.J., Gertz S.D., Gimple L.W., Owen R.M., Powers E.R., Roberts W.C. Effectiveness of recombinant desulphatohirudin (CGP 39393) in reducing restenosis after balloon angioplasty of atherosclerotic femoral arteries in rabbits. Circulation (1991) 84:232–243.[Abstract/Free Full Text]
  26. Gertz S.D., Gimple L.W., Ragosta M., Roberts W.C., Haber H.L., Powers E.R., Perez L.S., Sarembock I.J. Response of femoral arteries of cholesterol fed rabbits to balloon angioplasty with or without laser: emphasis on the distribution of foam cells. Exp Mol Pathol (1993) 59:225–243.[CrossRef][Web of Science][Medline]
  27. Gimple L.W., Gertz S.D., Haber H.L., Powers E.R., Roberts W.C., Sarembock I.J. Effect of intramural or chronic subcutaneous administration of heparin on femoral artery restenosis following balloon angioplasty in rabbits: a quantitative angiographic and histopathologic study. Circulation (1992) 86:1535–1546.
  28. Movat H. Demonstration of all connective tissue elements in a single section. Arch Pathol Lab Med (1955) 60:289–295.
  29. Stadius M.L., Gown A.M., Kernoff R., Collins C.L. Cell proliferation after balloon injury of iliac arteries in the cholesterol-fed New Zealand White rabbit. Arterioscler Thromb (1994) 14:727–733.[Abstract/Free Full Text]
  30. Casscells W. Migration of smooth muscle and endothelial cells: Critical events in restenosis. Circulation (1992) 86:723–729.[Free Full Text]
  31. Galis Z.S., Sukhova G.K., Lark M.W., Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest (1994) 94:2493–2503.[Web of Science][Medline]
  32. Galis Z.S., Sukhova G.K., Kranzhofer R., Clark S., Libby P. Macrophage foam cells from experimental atheroma constitutively produce matrix-degrading proteinases. Proc Natl Acad Sci (USA) (1995) 92:402–406.[Abstract/Free Full Text]
  33. Libby P., Galis Z.S. Cytokines regulate genes involved in atherogenesis. Ann NY Acad Sci (1995) 748:158–170.[Web of Science][Medline]
  34. Isner J.M., Kearney M., Bortman S., Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation. (1995) 91:2703–2711.[Abstract/Free Full Text]
  35. Holifield B., Helgason T., Jemelka S., Taylor A., Navran S., Allen J., Seidel C. Differentiated vascular myocytes: are they involved in neointimal formation? J Clin Invest (1996) 97:814–825.[Web of Science][Medline]
  36. Tanaka H., Suzuki A., Schwartz D., Sukhova G., Libby P. Activation of smooth muscle and endothelial cells following balloon injury. Ann NY Acad Sci (1995) 748:526–529.[Web of Science][Medline]
  37. London S.M., Mayberg S.M. Kinetics of bromodeoxyuridine uptake by smooth muscle cells after arterial injury. J Vasc Res (1994) 31:247–255.[Web of Science][Medline]

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