Skip Navigation

Cardiovascular Research 2000 47(4):759-768; doi:10.1016/S0008-6363(00)00120-6
© 2000 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Ohlstein, E. H.
Right arrow Articles by Chandra, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohlstein, E. H.
Right arrow Articles by Chandra, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2000, European Society of Cardiology

Application of in vivo and ex vivo magnetic resonance imaging for evaluation of tranilast on neointima formation following balloon angioplasty of the rat carotid artery

Eliot H. Ohlsteina,*, Anne M. Romanica, Lynne V. Clarka, Rasesh D. Kapadiab, Susanta K. Sarkarb, Robert Gagnonc and Sudeep Chandrab

aDepartment of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406-0939, USA
bDepartment of Physical and Structural Chemistry, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406-0939, USA
cStatistical Sciences, SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406-0939, USA

* Corresponding author. Tel.: +1-610-270-6071; fax: +1-610-270-6206 ohlstein{at}sbphrd.com

Received 28 October 1999; accepted 27 April 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Recent studies suggest that tranilast inhibits a variety of agents implicated in neointimal growth and restenosis in experimental animal models and humans. We report here a study evaluating the efficacy of tranilast in the rat carotid artery balloon angioplasty model, a model that mimics many aspects of the percutaneous transluminal angioplasty procedure in humans. Efficacy was determined based on in vivo and ex vivo magnetic resonance imaging (MRI) as well as by histomorphometry. The utility of this study, using a reverse paradigm, is to investigate if agents successful in the clinic can demonstrate efficacy in this animal model primary screen as measured by MRI and histomorphometry. Methods: Tranilast (300 mg/kg/day, p.o.) was administered to Sprague–Dawley rats 3 days prior to balloon injury and continued for 14 days after injury. Three methods of measuring the vascular injury that occurs in this model were employed: (1) in vivo MRI, used to measure in vivo lumen volumes for the carotid artery once at baseline (pre-surgery) and again at 14 days post angioplasty; (2) ex vivo MRI (and histomorphometry), used to evaluate the total arterial wall thickness and the intima-to-media ratio; and (3) analysis of collagen density, used to evaluate the efficacy of tranilast to abrogate collagen synthesis and deposition following vascular injury. Results: Tranilast provided 33% protection (P<0.05) from angioplasty-induced lumen narrowing as measured by MRI in vivo. The results of the ex vivo MR analysis of total wall thickness showed a 14% protection of angioplasty-induced narrowing (P<0.05), and the mean intima-to-media ratio showed a 39% (P<0.006) protection for the tranilast-treated rats. Histological analysis of the mean intima-to-media ratio demonstrated that tranilast provided 36% (P<0.01) protection in the intima-to-media ratio. Further, treatment with tranilast showed a 52% reduction in collagen density of the intimal layer and a 70% reduction in collagen density of the medial layer of the injured arteries. Conclusion: The data obtained by in vivo MRI, ex vivo MRI, histology and collagen analysis demonstrate that tranilast provided significant beneficial effects in inhibiting neointimal formation in the rat carotid artery model. Also this study, to the best of our knowledge, is the first to harness complimentary information from various technologies, including lumen patency by in vivo MRI, neointimal formation by ex vivo MRI and conventional histomorphometry, and histological analysis for collagen density, to provide a comprehensive understanding of the pathology in this disease model.

KEYWORDS Angioplasty; Extracellular matrix; Histo(patho)logy; NMR


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Percutaneous transluminal coronary angioplasty (PTCA) is an increasingly used procedure for treating patients with ischemic coronary artery disease; however, approximately 30–50% of patients suffer from vascular restenosis within 6 months of the procedure. Four principal cellular events have been identified contributing to lesion formation: (1) thrombosis, (2) cellular proliferation, (3) cellular migration and infiltration and (4) matrix production [1].

Existing evidence suggests that tranilast, an anti-allergic drug, may prevent restenosis after PTCA by inhibitory effects on proliferation, migration, c-myc gene expression, and collagen synthesis of vascular smooth muscle cells [2]. Several studies demonstrate that tranilast can inhibit collagen synthesis and platelet derived growth factor (PDGF) induced proliferation of vascular smooth muscle cells [3–5]. Tranilast has also been shown to stabilize mast cells [6], act as an antifibrotic agent [7], enhance nitric oxide (NO) production [8], and inhibit reactive oxygen species production [9], transforming growth factor β1 (TGF-β1) expression [10], prostaglandin E2 (PGE2) release [10], and endothelin expression [11]. All of these factors and mechanisms have been implicated in the pathogenesis of restenosis. A recent study in the rabbit reported that tranilast decreased intimal area, intima:media ratio, stenosis ratio and vascular DNA content after endothelial injury [12]. Following photochemically induced thrombosis, tranilast also produced dose-dependent inhibition of intimal thickening in rat femoral arteries [13]. While a large randomized clinical trial to test the efficacy of tranilast is underway, in several recent smaller clinical trials in humans, tranilast was reported to be efficacious in preventing restenosis following PTCA [14–16]. Based on these findings, we examined the effects of tranilast in the rat carotid artery balloon angioplasty model. This is the experimental animal model most extensively used as a primary screen for evaluating agents that can inhibit vascular damage and neointimal formation. The utility of this study, using a reverse paradigm, is to investigate if agents successful in the clinic can demonstrate efficacy and validate this primary screen. Furthermore, the efficacy of tranilast in other pre-clinical models has only been measured by conventional histomorphometry which is limited from addressing the vasoconstrictive aspect of the disease in vivo due to the inherent problems associated with absence of local internal blood pressure and tissue shrinkage [17,18]. Recently magnetic resonance imaging (MRI) methods have been successfully incorporated to address this aspect of the disease [17–19]. The present study uses MRI to perform a comprehensive investigation of tranilast in the rat carotid model. In addition to in vivo MRI and conventional histology, ex vivo high resolution MRI was used to cross validate the histomorphometric measurements. Further, collagen analysis was also performed to demonstrate the efficacy of tranilast at reducing collagen deposition in this model, given its increased fibrillar collagen content.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Balloon angioplasty
Male, Sprague–Dawley rats (350–400 g) were pretreated daily by oral gavage with either the vehicle (0.5% methyl cellulose) or tranilast (300 mg/kg, p.o.) for 3 days prior to surgery. The dose of tranilast used for these studies was based on previous reports [12]. Further, based on DMPK analysis, this dose of tranilast achieved blood levels comparable to those seen in patients. In patients, studies have been conducted in which the total daily dose was 600–900 mg. Before surgery the rats were anesthetized with a combination of ketamine (50 mg/kg) and xylazine (10 mg/kg, i.p.). Left common carotid artery balloon angioplasty was performed under aseptic conditions as described previously [20]. The left distal common carotid and the external carotid arteries were exposed through a midline incision in the neck. A sterile, 2F Fogarty arterial embolectomy catheter (model 12-060-2F, Baxter Healthcare) was introduced through the external carotid artery and guided through the common carotid artery down to the aortic arch. The balloon was then distended sufficiently with saline to generate slight resistance and withdrawn back to the site of insertion. This procedure was performed twice. The catheter was subsequently removed and the external carotid ligated with 4.0 silk suture without occluding flow to the occipital artery. The wound was closed with 3-0 Prolene suture (Ethicon, Somerville, NJ, USA), and the area was swabbed with betadine solution. The animals were housed in Plexiglass cages under a 12 h light–dark cycle. All animals were allowed food and water ad libitum post surgery. The animal study protocol was approved by the SmithKline Beecham animal care committee. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 In vivo MRI methods
Rats from each of the two groups (n=14 in vehicle- and n=13 in drug-treated group) were imaged twice, once at baseline and again at day 14 post angioplasty, using a 4.7T/40 cm Bruker imaging spectrometer (Billerica, MA, USA). For each imaging session the animals were anesthetized lightly with a 1–1.5% isoflurane (Ohmeda Caribe) in 0.8–1.0/min of O2. A 2-D Fourier transform multislice spin-echo sequence (TR/TE=4000/23 ms; Field of view=3x3 cm; matrix size=256x256; slice thickness=1.5 mm; no. of slices=18) was used with flow dephasing gradients (1G/cm; duration=5 ms). A 9 strut half-birdcage RF resonator was used for both transmission and reception. All images were cardiac gated with the trigger for data acquisition set to 40 ms from the QRS complex. Motion artifacts from respiration did not significantly affect the image quality eliminating the requirement for simultaneous respiratory gating. Data acquisition time was {approx}35 min per animal.

The index for quantitative monitoring of the vascular injury was defined as the lumen volume measured along a fixed section of the carotid artery. This volume was obtained by adding cross-sectional lumen areas from contiguous slices over a length of 1.05 cm and multiplying the sum so obtained by the thickness of each slice. The origin of this length was always set with respect to internal anatomical landmarks (3 mm distal to the external and internal carotid bifurcation) so that minor positioning variations in the magnet would not contribute to significant errors. The first two slices from the bifurcation (each 1.5 mm thick) were excluded from quantitative measurements since no apparent changes in the lumen areas were observed at these sites in the MRI for both the control and the drug-treated groups.

2.3 Ex vivo MRI methods
At the end of the study, the animals were euthanized (sodium pentobarbital; 65 mg/kg, i.p.) and their common carotid arteries were dissected following in situ perfusion fixation (with saline and formalin at a pressure of 100 mmHg). The carotid arteries were cleared of all external tissue and stored in 10% buffered formalin. For ex vivo MRI, the arteries were inserted in a capillary tube (2.2 mm diameter) and then centered in a 6 mm RF coil. A 3-D Fourier Transform spin-echo sequence (TR/TE=500 ms/6 ms; matrix size=256x128x64; field of view of 0.4x0.4x2.0 cm providing a resolution of 16x32x312 µm [3]) on a Bruker (Billerica) 9.4T vertical bore magnet. Data acquisition time was {approx}135 min per vessel.

Total wall thickness (intima+media) was measured by a combination of manual/intensity based segmentation at four sites chosen to maximally overlap sections used in conventional histology (2.5, 4.5, 6.5 and 8.5 mm from the anatomical bifurcation of the internal and external carotid arteries). In the absence of a clear demarcation between media and neointimal layers in the ex vivo MR images, the initmal area were obtained by subtracting the area of normal vessels without the intimal layer (contralateral carotid arteries of the same animals) from the total vessel wall (of the injured artery). The I/M ratios were calculated using the intimal area so obtained.

2.4 Histological methods
Following ex vivo analysis, four 5-µm arterial cross-sections were cut from paraffin blocks containing the middle portion of these arteries. Four sections of the artery were averaged for the histological analysis. Section 1 corresponds to the section closest to the middle of the intact artery and section 4 corresponds to the portion of the artery most proximal to the aortic arch end of the vessel. The arteries were then processed for standard hematoxylin and eosin staining. The morphometry of these vessels was quantified using a Bioscan Optimus (Edmonds, WA, USA) cell imaging system. For histomorphometric analysis, the overall area of the vessel, the overall area under the elastic lamina, and area of the lumen were determined for each section of the vessel from which appropriate algebraic calculations were made to obtain the neointima, media and lumen area for each vessel.

2.5 Measurement of percent collagen content in blood vessels
Carotid artery tissue samples were collected 14 days after angioplasty and prepared for staining as described above (histological methods). Following standard histological processing and embedding in paraffin, 6 µm-thick sections were prepared for collagen analysis using picrosirius red staining. Briefly, sections were stained with 0.1% sirius red FB3A (Polysciences, Warrington, PA, USA) in saturated picric acid (Sigma, St. Louis, MO, USA) for 30 min at room temperature. The stained sections were observed under polarized light and viewed using an Olympus Ix70 microscope. With this method, collagen is stained orange/red. To determine the percentage of fibrillar collagen content in the blood vessels, image analysis of the stained sections was performed using a digital color threshold routine in Optimus® (Norwood, MA, USA). The percentage collagen content was calculated relative to the total vessel area. Collagen analysis was performed for the intima and media of balloon-injured vessels of animals treated with tranilast or vehicle (n=3 animals, two sections analyzed for each) and for a sham group (n=3 animals, two sections analyzed for each).

2.6 Data analyses and statistics
All results (in vivo MRI, ex vivo MRI and histomorphometry) are presented as mean±SEM and n represents the number of animals used in a particular group. Image analysis was performed without a priori knowledge of treatment. Quantitative data from MR images were sorted according to treatment after completion of data analysis. For trained observers, both inter- and intra-observer errors were typically of the order of 5% in calculated lumen volumes. Statistical comparisons were made using either a one-way analysis of variance, or a t-test. Values were considered to be significant at P<0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 MRI analysis — in vivo
Fig. 1 shows a representative in vivo MR image slice obtained with an in-plane resolution of 120x120 µms and a slice thickness of 1.5 mm. Fig. 2 shows the images acquired serially from one representative animal of each group at baseline and at day 14. In each case, anatomically near-identical areas are shown to demonstrate the efficacy of tranilast to provide protection from lumen loss after angioplasty. The volume index, calculated from seven such contiguous slices, was plotted for both the vehicle- and the tranilast-treated groups at pre-surgery and at day 14 post surgery (Fig. 3). In terms of absolute values, the baseline presurgery value for the tranilast-treated group was 10.12±0.26 mm3 for the right artery and 10.18±0.27 mm3 for the left artery. The corresponding values at baseline for the vehicle-treated group were 9.90±0.25 and 9.92±0.21 mm3, respectively. On day 14, the left artery had a volume of 7.53±0.62 mm3 for the tranilast-treated group and 5.9±0.59 mm3 for the vehicle-treated group. The corresponding values for the right arteries were 11.82±0.29 and 11.58±0.24 mm3, respectively at this time point. As indicated in Fig. 3, the left artery for both groups had developed statistically significantly smaller lumen volumes on day 14 as compared to their baseline values (P<0.005 for the vehicle group; P<0.005 for the tranilast group). Taken as a percentage of the baseline values however, the vehicle group demonstrated a 40.4% decrease in lumen volume as measured by this index as opposed to 26% for the tranilast-treated group. Therefore, tranilast provided a 33% protection in terms of reduction of in vivo lumen volume. Interestingly, a comparison of the right arteries in both groups indicated a compensation for the decrease in lumen of the left artery as evidenced by their statistically significant increased lumen volumes on day 14 with respect to their baseline volumes (P<0.05 for the vehicle group; P<0.05 for the drug group).


Figure 1
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Representative in vivo MR image of a single slice of a rat neck region with in-plane resolution of 120x120 µms and a slice thickness of 1.5 mm. The two carotid arteries can be clearly seen on both sides of the central tracheal tract (see arrows). The lumen cross-sectional area can be reproducibly and consistently measured from such images. Seven such slices were used to arrive at an in vivo index for the lumen volume for each artery of each animal at each time point.

 

Figure 2
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Images of the ballooned artery from one representative animal taken from each group. The images are taken from the same animal from nearly identical anatomical locations at two time points, as indicated. On day 14, Tranilast provided significantly larger protection from lumen loss as compared to the vehicle-treated group (P<0.05).

 

Figure 3
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Bar graph showing the in vivo MR volume index calculated from the MR images (as shown in Fig. 1) plotted against the two time points of measurements. Animals in the drug group were treated with 300 mg/kg tranilast p.o.; the vehicle group received 0.5% methyl cellulose. Numbers are plotted as mean±SEM; n=13 for drug group and n=23 for the vehicles. Solid bars indicate the right uninjured arteries and open bars indicate the left injured arteries.

 
3.2 MRI analysis — ex vivo
Fig. 4 demonstrates cross-sectional views of the rat carotid arteries under different experimental conditions. The ex vivo MR images of normal vessels (media) show an iso-intense ring surrounded irregularly by adventitia (Fig. 4A). The boundary between the media and adventitia is demarcated by a small dark band, representative of the innermost layer of adventitial tissue. The image of ballooned artery (vehicle-treated) shows a significant increase in wall thickness (Fig. 4B). An image from a vessel of a rat treated with tranilast is shown in Fig. 4C. In conventional histomorphometric analysis, significant attenuation of the intima/media ratio was obtained (Fig. 5). By ex vivo MR analysis, there was significant attenuation of neointimal growth as can be seen by the reduction in the total wall thickness of the artery in the tranilast treated group (Fig. 6a). The mean wall thickness area of the four sections from the various groups for MRI analysis were 0.279 ±0.012 mm2 in the vehicle-treated animals and 0.239±0.017 mm2 in the drug-treated animals (Fig. 6a). The measurements made from conventional histology were 0.234±.020 mm2 in the vehicle-treated animals and 0.226±0.013 mm2 in the drug-treated animals (Fig. 6a). Neointima/media ratios were calculated from MR (vehicle-treated=1.143±0.091; drug-treated=0.693±0.122; P<0.006) and histology (vehicle-treated=1.054±0.082; drug-treated=0.679±0.117; P<0.012) and are shown in Fig. 6b. The data from the MR and histomorphometry show significant protection in the vessels from rats treated with tranilast.


Figure 4
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Representative ex vivo MR images of (a) a normal, rat carotid artery; (b) a balloon vehicle-treated and (c) tranilast-treated. The non-circular nature of the soft deformable arteries, as is often seen in ex vivo methods, is mainly due to lack of internal blood pressure and flow. This often hinders measurement of a relevant cross-sectional lumen analysis by ex vivo tools. The field of view is 0.4x0.4 mm.

 

Figure 5
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Bar graph showing the histological effect of tranilast on the media area, neointimal area, and neointima/media ratio 14 days following endothelial injury in the rat carotid artery. The shaded bars are the vehicle-treated group, and the cross-hatched bars are the tranilast-treated group. Results are presented as mean±SEM (n=13 for drug-treated group and n=23 for the vehicle-treated group). The asterisk indicated significant differences between the two groups (P<0.05).

 

Figure 6
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 (a) Bar graph showing the comparison of total wall area measured with MRI and histology. Results are presented as mean±SEM. The MRI results are P<0.0326 (drug-treated group n=12, represented by cross-hatched bar; vehicle-treated group n=21, filled bar), compared to conventional histology results (P<0.927, drug-treated group n=13, represented by striped bar, vehicle-treated n=21, open bar.). The differences could be attributed to differences in analysis procedures between MR and histology data. (b) Bar graph comparing I/M ratios as calculated from MRI and histology. The MR results were P<0.006 (vehicle-treated n=21 represented by cross-hatched bar, vs. drug-treated n=12, filled bar), compared to histology results P<0.012 (represented by striped bar, vehicle-treated n=21, drug treated n=13 open bar). **, P<0.01 *, P<0.05. The differences in the P values could be attributed to differences in analysis procedures between MR and histology data.

 
3.3 Histological analysis
A pronounced neointima was formed in the left common carotid artery 14 days following angioplasty (Fig. 4). No neointima was detected in the contralateral arteries in the drug or vehicle-treated rats. Administration of tranilast produced a significant reduction in the mean neointima to media ratio formation compared to vehicle-treated rats (36%) (Fig. 5). To explore the correlation between the in vivo MRI data and conventional histology, the MR index was plotted against the corresponding I/M ratio index for each individual animal in the study (Fig. 7a). The plot indicates the large inter-animal variability with respect to both indices and illustrates a statistically significant degree of correlation between the two indices (correlation coefficient r=–0.54 for the vehicle group (n=23), P<0.05; r=–0.75; for the drug-treated group (n=13), P<0.05). Additionally, the correlation plot between ex vivo MRI and histomorphometry is also shown in Fig. 7b. The data indicate a significant positive correlation between the measurements (correlation coefficient r=0.71 for the vehicle group (n=20) with P<0.05; and r=0.89 for the drug-treated group (n=12) with P<0.05).


Figure 7
View larger version (11K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 (a) In vivo MR volume index plotted against the ex vivo histomorphometric (average I/M ratio) index. The correlation between the two indices is significant but not very high (r=–0.54 for the vehicle-treated group (n=23); r=–0.75 for the drug-treated group (n=13); P<0.05). This plot delineates the large inter-animal variability that exists in this model; The global values are plotted as mean±SEM. The squares represent individual drug-treated animals (open) as well as the overall drug-treated global mean (filled). The circles represent the individual vehicle-treated animals (open) and the mean values (filled). (b) Average I/M ratio obtained from ex vivo MRI was plotted against the histomorphometric (average I/M ratio) index over four sections per artery. The two indices are significantly correlated with relatively positive correlation coefficients (r=0.71 and r=0.88 for vehicle (n=20) and drug group (n=12), respectively; P<0.05). The global values are plotted as mean±SEM. The squares represent individual drug-treated animals (open) as well as the overall global mean for the same group (filled). The circles (open) represent individual vehicle-treated animals and their global mean (filled).

 
3.4 Percent collagen content analysis
Tissue sections of balloon-injured carotid arteries collected 14 days after angioplasty were analyzed for percent collagen content following picrosirius red histological staining (Fig. 8A). The results demonstrated that the average collagen content within injured blood vessels treated with vehicle (n=3) was 2.7±0.5% in the intima and 7.1±0.6% in the media (Fig. 8B). In animals treated with tranilast (300 mg/kg, n=3), the collagen content was 1.3±0.2% in the intima and 2.2±0.2% in the media (Fig. 8B). Tranilast reduced the collagen content in the intima after balloon angioplasty by 52% (P<0.02) and in the media by 70% (P<0.005).


Figure 8
View larger version (68K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 (A) Histological analysis of carotid artery vessels for collagen content by picrosirius red staining. Uninjured control (original magnification 50x, A). Tranilast-treated sample collected 14 days after angioplasty (original magnification 50x B). Vehicle-treated sample collected 14 days after angioplasty (original magnification 50x, C). Collagen is typically stained orange/red. (B) Quantitative analysis of the percent collagen content in carotid arteries treated with tranilast or vehicle. Image analysis of the stained sections was performed using a digital color threshold routine in Optimus®. Collagen analysis was performed for the intima and media of balloon-injured vessels of animals treated with tranilast or vehicle (n=3 animals analyzed for each condition and two sections from each animal) and collected 14 days after angioplasty. *, P<0.02, **, P<0.005. Open bars, vehicle treated. Solid bars, tranilast treated.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
Previous studies indicate that tranilast inhibits vascular smooth muscle cell migration and proliferation as well as accumulation of extracellular matrix [3–5,21]. Since various growth factors and cytokines are involved in matrix synthesis as well as inflammatory responses, the rat carotid artery balloon injury model is an ideal experimental model for studying the efficacy of agents that potentially inhibit neointimal growth. The present study is in agreement with previous reports that tranilast may prevent restenosis after PTCA and extends those findings to the rat balloon-injured carotid artery model. Notably, demonstration of positive correlation of results generated in the rat balloon-injury model with those generated in clinical trials is of great importance since the rat balloon-injury model is the most commonly used animal model for restenosis. Positive correlations between clinical outcome and experimental laboratory studies, using diagnostic tools (e.g. MRI) commonly used in the clinic, improves the confidence of results generated in the laboratory.

The in vivo MRI data is best appreciated when viewed as additional information obtained along with the ex vivo measurements. As a non-invasive measurement of the disease in its most native in vivo state, MRI clearly adds an extra dimension to the study. The lumen volume index obtained with in vivo MRI provides the opportunity to understand the in vivo injury-response following balloon denudation in the rat and generates additional information regarding actions of pharmacological agents under the influence of numerous in vivo parameters. It has been postulated therefore [18,19], that the efficacy of a therapeutic agent as measured by these two indices (in vivo lumen index from MRI and ex vivo I/M ratio from histomorphometry) may not be identical. Additionally, systematic variations in the two technologies, e.g. spatial volume effects from thicker sections in in vivo MRI, shrinkage artifacts in histology, etc. may adversely affect the correlation. To explore further this issue, a correlation between the MR index and the corresponding I/M ratio index was determined. As shown in Fig. 7A, there was a statistically significant correlation between the measurements by the two techniques. Further, the difference between the global mean values of the two measurements is more pronounced on the histology axis than on the MR axis. These data, therefore, add further merit to the hypothesis that despite a significant correlation between structural regrowth of the intimal layer and lumen loss, there may be compensatory mechanisms (vascular or functional or both) of the disease in vivo, which may influence the in vivo measurements leading to smaller differences in the MR volume index between the treated and untreated groups of animals [18,19]. This further highlights the importance of in vivo MRI since it demonstrates that a large reduction of I/M ratio may not always be accompanied by a concomitant increase in in vivo lumen volume or area. In clinical evaluations, changes in in vivo lumen diameters, assessed by angiographic techniques usually serve as clinical end points. From this perspective, use of in vivo MRI provides an option to evaluate such in vivo indices in early pre-clinical testing [18,19]. Certainly in experimental models where arterial remodeling is important, like the rabbit, an in vivo index could provide critical information [22]. Moreover, additional information generated by in vivo MRI volumetric analysis provides an opportunity to study the changes in lumen volume at various time points in the whole animal, an option not feasible with conventional histological analysis [19]. In this context it is important to note that in vivo MRI data has equal contributions from all relevant slices chosen from appropriate sections on the artery. Therefore, this index represents an appropriate metric for the global injury along the length of the artery. A secondary advantage of calculating volumes comes from the fact that random errors from tracing, thresholding, etc. at each slice does not significantly affect the data. Therefore the volume index, measured over appropriate sections, is more robust than a single slice measurement. The resolution of the in vivo MR images used in this analysis was of the order of 120 µm in-plane. In this experimental set up it would be reasonable to assume that a change of 2–3 pixels in-plane in lumen diameters would be discernible, with some image analysis experience.

The mean overall wall area of the vessels, as measured by ex vivo MRI, in the tranilast-treated group was significantly lower than that in the vehicle-treated group. By histomorphometrical analysis, though, no difference on total wall area were noted between the tranilast-treated group and the vehicle-treated group. Some systemic variables like exposure to repeated dehydration/rehydration cycles, differential sensitivity to hydrated molecules like collagen, may contribute to such changes when absolute areas are compared.

There is a close agreement in the inhibition of neointima formation by tranilast as calculated by the mean intima-to-media ratio by ex vivo MRI and conventional histology. In addition, a significant correlation exists between the ex vivo MR estimates of I/M ratios and those obtained from histology confirming the fact that the two technologies measure comparable aspects of the disease. Mathematically the dynamic range of the changes in neointima-to-media ratio is higher than that in the total wall area. Tranilast achieved significant protection for the I/M ratio index in both ex vivo MR and histology. For the total wall area measurements, however, it achieved significant protection only in ex vivo MRI.

Collagen deposition contributes to neointima formation following balloon injury [1,12]. It has also been suggested that increased collagen production contributes to smooth muscle cell migration [3]. Tranilast has been reported to inhibit collagen synthesis in cultured human vascular smooth muscle cells [3,5] and in rabbit balloon injury model [12]. The results of the studies presented here directly demonstrate that tranilast reduces percent collagen content in a rat model for restenosis and further support that reduction in collagen content directly correlates with reduced intimal thickening. However, it has been reported by Coats et al. [23] that, following balloon angioplasty in a rabbit model there is an increase in total collagen content although restenotic vessels contain less collagen than nonrestenotic vessels. This reduced collagen content correlated with an observed increase in matrix metalloproteinase (MMP) activity. The reorganization of the extracellular matrix that occurs following balloon angioplasty is due in part to enhanced collagen and MMP synthesis. Therefore the balance between matrix deposition and degradation influences the degree of restenosis. However, in the rat model used in our study, less MMP activity is present compared to the rabbit model used by Coats et al. [23]. In particular, collagenase is absent in the rat carotid injury model [24]. Since collagenase is not expressed in the rat model, then the overall levels of fibrillar collagen are expected to be greater. This would explain why an increased fibrillar collagen content in the rat model of neointima formation when compared to the rabbit model. These results demonstrate clearly that in the animals treated with tranilast, collagen content was reduced, and this reduction corresponded with increased lumen patency and reduced neointima formation.

In summary, the data obtained by in vivo, ex vivo MRI and histology demonstrated that tranilast provided beneficial effects in inhibiting neointimal formation in the rat carotid artery balloon injury model. Also this study, to the best of our knowledge, is the first to harness complimentary information from various technologies i.e. lumen patency by in vivo MRI, neointimal formation by ex vivo MRI morphometry and conventional histomorphometry, and collagen analysis to provide a comprehensive analysis of neointimal formation in this experimental model.

Time for primary review 30 days.


   Acknowledgements
 
The authors would like to acknowledge Dr. C. Louden and Ms. R. Mirabile for histomorphometric analysis; Mr. Joshua Levine, Mr. David Arcangelo and members of the rodent research group for excellent technical assistance in MRI data acquisition and analysis. The authors also thank Ms. Cynthia Burns for technical assistance in collagen staining and analysis.


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

  1. Douglas S.A. Coronary restenosis. Feuerstein G.Z., ed. (1996) New York: Marcel Dekker. 269–322.
  2. Koda A., Nagai H., Watanabe Y. Inhibition of hypersensitivity reactions by a new drug, N(3'4'-dimethoxycinnamoyl) anthranilic acid (N-5'). J Allergy Clin Immunol (1976) 57:396.[CrossRef][Web of Science][Medline]
  3. Miyazawa K., Kikuchi S., Fukuyama J., Hamano S., Ujiie A. Inhibition of PDGF-, TGF-B1-induced collagen synthesis, migration, and proliferation in vascular smooth muscle cells from spontaneously hypertensive rats. Atherosclerosis (1995) 118:213–221.[CrossRef][Web of Science][Medline]
  4. Miyazawa K.J., Hamano S., Ujiie A. Antiproliferative and c-myc mRNA suppressive effects of tranilast on newborn human vascular smooth muscle cells in culture. Br. J. Pharm (1996) 118:915–922.[Web of Science][Medline]
  5. Tanaka K., Honda M., Kuuramochi T., Morioka S. Prominent inhibitory effects of tranilast on migration and proliferation of and collagen synthesis by smooth muscle cells. Atherosclerosis (1994) 107:179.[CrossRef][Web of Science][Medline]
  6. Suzuki H., Miura S., Liu Y., Tsuchiya M., Ishii H. Substance P induces degranulation of mast cells and leukocyte adhesion to venular endothelium. Peptides (1995) 16:1447–1452.[CrossRef][Web of Science][Medline]
  7. Yamada H., Ide A., Sugiura M., Tajima S. Treatment of cutaneous sarcoidosis with tranilast. J Dermatol (1995) 22:149–152.[Medline]
  8. Hishikawa K., Nakaki T., Hirahashi J., Marumo T., Saruta T. Tranilast restores cytokine-induced nitric oxide production against platelet-derived growth factor in vascular smooth muscle cells. J Cardiovasc Pharmacol (1996) 28:200–207.[CrossRef][Web of Science][Medline]
  9. Miyachi Y., Imamura S., Niwa Y. The effect of tranilast of the generation of reactive oxygen species. J Pharmacobio-Dyn. (1987) 10:255–259.[Medline]
  10. Suzawa, H. Kikuchi, S. Ichikawa, K. Koda, A. Inhibitory action of tranilast, an anti-allergic drug, on the release of cytokines and PGE2 from human monocytes–macrophages. 1992, 60:85–90.
  11. Nagai H., Suda H., Kitagaki K., Koda A. Effect of tranilast on endothelin-induced bronchoconstriction in guinea pigs. J. Pharmacobio-Dyn. (1991) 14:309–314.[Medline]
  12. Fukuyama J., Ichikawa K., Miyazawa K., Hamano S., Shibata N., Ujiie A. Tranilast suppresses intimal hyperplasia in the balloon injury model and cuff treatment model in rabbits. Jpn. J. Pharmacol (1996) 70:321–327.[Medline]
  13. Kikuchi S., Umemura K., Kondo K., Nakashima M. Tranilast suppresses intimal hyperplasia after photochemical induced endothelial injury in the rat. Eur J Pharm (1996) 295:221–227.[CrossRef][Web of Science][Medline]
  14. Holmes D., Fitzgerald P., Goldberg S., et al. The PRESTO (prevention of restenosis with tranilast and its outcomes) protocol: A double-blind, placebo-controlled trial. Am Heart J (2000) 139:23–31.[Web of Science][Medline]
  15. The Treat Study Investigators. The impact of tranilast on restenosis following coronary angioplasty the tranilast restenosis following angioplasty trial (TREAT). Circulation (1994) 90:I–652.
  16. Kosuga K., Tamai H., Ueda K., et al. Effectiveness of tranilast on restenosis after directional coronary atherectomy. Am. Heart J (1997) 134:712–718.[CrossRef][Web of Science][Medline]
  17. Summers R.M., Hedlund L.W., Cofer G.P., Gottsman M.B., Manibo J.F., Johnson G.A.M.R. Microscopy of the rat carotid artery after balloon injury by using an implanted imaging coil. Magn. Reson. Med. (1995) 33:321–323.
  18. Cook N.S., Zerwes H.G., Pally C., Rudin M., Hof R.P. Spirapril and Cilazapril inhibit neointimal lesion development but cause no detectable inhibition of lumen narrowing after carotid artery balloon catheter injury in the rat. Blood Pressure (1993) 2:322–331.[Medline]
  19. Chandra S., Vickery-Clark L., Coatney R.W., Phan L., Sarkar S.K., Ohlstein E.H. Application of serial in vivo magnetic resonance imaging to evaluate the efficacy of endothelin receptor antagonist SB 217242 in the rat carotid artery model of neointima formation. Circulation (1998) 97:2252–2258.[Abstract/Free Full Text]
  20. Douglas S.A., Louden C., Vickery-Clark L.M., et al. A role for endogenous endothelin-1 in neointimal formation after rat carotid artery balloon angioplasty. Circ. Res. (1994) 75:190–197.[Abstract/Free Full Text]
  21. Miyazawa K., Fukuyama J., Misawa K., Hamano S., Ujiie A. Tranilast antagonizes angiotensin II and inhibits its biological effects in vascular smooth muscle cells. Atherosclerosis (1996) 121:167–173.[CrossRef][Web of Science][Medline]
  22. Kakuta T., Currier J.W., Haudenschild C.C., Ryan T.J., Faxon D.P. Differences in compensatory vessel enlargement, not intimal formation accounts for restenosis after angioplasty hypercholesteromic rabbit model. Circulation (1994) 89:2809–2815.[Abstract/Free Full Text]
  23. Coats W.D. Jr., Whittaker P., Cheung D., Currier J.W., Han B., Faxon D.P. Collagen content is significantly lower in restenotic versus nonrestenotic vessels after balloon angioplasty in the atherosclerotic rabbit model. Circulation (1997) 95:1293–1300.[Abstract/Free Full Text]
  24. Bendeck M.P., Zempo N., Clowes A.W., Galardy R.E., Reidy M.A. Smooth muscle cell migration and matrix metalloproteinase expression after arterial injury in the rat. Circ Res (1994) 75:539–545.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Ohlstein, E. H.
Right arrow Articles by Chandra, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ohlstein, E. H.
Right arrow Articles by Chandra, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?