Author + information
- Received February 21, 2008
- Revision received August 26, 2008
- Accepted September 4, 2008
- Published online March 31, 2009.
- Ronen Jaffe, MD*,†,§,
- General Leung, MSc‡,§,
- Nigel R. Munce, MSc‡,§,
- Amandeep S. Thind, BSc‡,§,
- Howard Leong-Poi, MD†,§,
- Kevan J.T. Anderson, BSc‡,§,
- Xiuling Qi, PhD, MD‡,
- Judy Trogadis, BSc†,
- Ariella Nadler, BA*,
- Davida Shiff*,
- Jamie Saperia*,
- Julia Lockwood*,
- Chaim Jacobs*,
- Beiping Qiang, MD*,†,
- Aaron Teitelbaum, MD, MSc*,
- Alexander J. Dick, MD*,§,
- John D. Sparkes, MSc*,
- Jagdish Butany, MD§∥,
- Graham A. Wright, PhD*,‡,§ and
- Bradley H. Strauss, MD, PhD*,§,* ()
- ↵*Reprint requests and correspondence:
Dr. Bradley H. Strauss, Reichmann Chair in Cardiovascular Sciences, Sunnybrook Health Sciences Centre, 2075 Bayview Avenue, Room A-253, Toronto, Ontario, Canada M4N 3M5
Objectives We sought to perform the first systematic study of the natural history of chronic total arterial occlusions (CTOs) in an experimental model.
Background Angioplasty of CTOs has low success rates. The structural and perfusion changes during CTO maturation, which may adversely affect angioplasty outcome, have not been systematically studied.
Methods Occlusions were created in 63 rabbit femoral arteries by thrombin injection. Histology, contrast-enhanced magnetic resonance imaging, relative blood volume (RBV) index, and micro-computed tomography imaging were analyzed at 2, 6, 12, and 18 to 24 weeks.
Results Early changes were characterized by an acute inflammatory response and negative arterial remodeling, with >70% reduction of arterial cross-sectional area (CSA) from 2 to 6 weeks. Intraluminal neovascularization of the CTO occurred with a 2-fold increase in total (media + intima) microvessel CSA from 2 to 6 weeks (0.014 ± 0.002 mm2to 0.023 ± 0.005 mm2, p = 0.0008) and a 3-fold increase in RBV index (5.1 ± 1.9% to 16.9 ± 2.7%, p = 0.0008). However at later time periods, there were significant reductions in both RBV (3.5 ± 1.1%, p < 0.0001) and total microvessel CSA (0.017 ± 0.002 mm2, p = 0.011). Micro-computed tomography imaging demonstrated a corkscrew-like recanalization channel at the proximal end at 6 weeks that regressed at later time points. These vascular changes were accompanied by a marked decrease in proteoglycans and accumulation of a collagen-enriched extracellular matrix, particularly at the entrance (“proximal fibrous cap”).
Conclusions This study is the first to systematically analyze compositional changes occurring during CTO maturation, which may underlie angioplasty failure. Negative remodeling, regression of intraluminal channels, and CTO perfusion, together with the accumulation of dense collagen, may represent important targets for novel therapeutic interventions.
Arterial chronic total occlusions (CTOs) in the coronary and peripheral vasculature are common and are associated with significant morbidity and adverse outcomes (1,2). Percutaneous angioplasty procedural success rates for CTOs are much lower than in stenosed-but-patent arteries, primarily due to failure of guidewire crossing through the occlusion (3–5), resulting in either referral of these patients for bypass surgery or avoidance of revascularization. Angioplasty success rates are inversely related to the age of the occlusion (1), suggesting that difficulties in crossing CTOs with guidewires are due to specific composition and physical characteristics of the CTO that change over time. To date, the pathological basis for angioplasty failure in CTOs has not been clearly defined. Only a small number of human CTO pathological specimens have been reported in the literature (6,7), which are limited by uncertain assessment of the age of the occlusion and inattention to specific geographic levels within the CTO. Neovascularization of the CTO lumen may constitute an important aspect of the maturation process. Previous studies of human arteries and experimental CTOs have suggested that the presence of microvessels within the CTO lumen may facilitate guidewire crossing, either as a direct pathway or by enhancing favorable changes in the composition of the surrounding extracellular matrix (6–8). Thus, the objective of this study was to perform an in-depth and detailed analysis of the maturational changes occurring within a nonatherosclerotic CTO model that is initiated by an acute thrombus formation. To accurately assess the spatial and temporal changes in the specific components, we used an approach that combined histology, specific matrix stains, and complementary in vivo and ex vivo imaging.
The occlusion model
Animal experiments conformed to the “Position of the American Heart Association on Research Animal Use.” Approval for experiments was obtained from St. Michael's and Sunnybrook Hospital Animal Care Committees. Arterial occlusions were created in 38 male New Zealand White rabbits (63 arteries, Charles River Canada, St. Constant, Quebec, Canada), weighing 3.0 to 3.5 kg, by injection of thrombin solution (100 IU, Millipore cat. no. 82-036-3, Kankakee, Illinois) into an isolated femoral artery segment, as previously described (9). Ligatures were maintained up to 60 min to ensure a persistent occlusion. Animals were then returned to their cage and fed a regular diet. Animals were sacrificed at 2, 6, 12, 18, and 24 weeks (range 12 to 20 CTOs/time point) after creation of the CTO. Because there were no quantitative or qualitative differences in any of the analyses between 18- and 24-week-old CTOs, the results of these 2 groups were combined. Magnetic resonance imaging (MRI) imaging was performed on the animals immediately before they were sacrificed, which was followed by ex vivo micro-computed tomography (CT) imaging and histological analysis.
We used MRI to quantify blood volumes within the CTO lumen as a measurement of functioning neovascular microvessels. The MRI studies were performed in 30 rabbits (30 arteries, 5 to 10 CTOs/time point) on a 3T GE Excite Scanner (GE Healthcare, Milwaukee, Wisconsin) by the use of a custom (∼5 × 3 cm) surface coil secured over the tissue around the occluded femoral artery. Occlusions were imaged with the use of an elliptic-centered fast spoiled gradient echo sequence (repetition time/echo time/θ = 8.3/2.1/30°, 31.25-kHz receive bandwidth, resolution of 0.25 mm in plane, 0.6 mm through plane). The elliptic ordering of acquisition procures most of the contrast during the first several seconds and, thus, the images are more representative of the contrast close to the start of the imaging.
T1-weighted images were obtained 16 s after the injection of 0.05 ml/kg Clariscan (Feruglose, GE Healthcare, Chalfont, United Kingdom). To maintain an adequate signal-to-noise ratio, each data point was consecutively averaged 4 times, yielding an imaging time of 216 s. Clariscan (Nycomed Imaging, Oslo, Norway) remains inside blood vessels and reflects relative blood volume (RBV) within the CTO lumen (10,11), serving as a surrogate for perfusion. Regions of interest were selected in the proximal part of the CTO on each of the T1-weighted MR images to calculate average signal intensity. Relative distribution volume of Clariscan was derived from the ratio of tissue signal intensity in the region of interest compared with the vessel segment immediately proximal to the CTO (12).
Intravenous heparin (3,000 U) was administered to the animals before they were sacrificed to prevent blood coagulation in the microvasculature. Microfil (30 ml, Flowtech, Carver, Massachusetts) was injected into the abdominal aorta at a pressure of 90 mm Hg, as measured by a handheld manometer. The femoral arteries were fixed in formalin for 48 h, embedded in 1% w/w agarose gel, and imaged in the micro-CT (MS-8, GE Medical Systems, London, Ontario). A 17-μm resolution was achieved (13). Three-dimensional cone beam CT datasets were acquired in 2.5 h with 905 views at 35 μm. X-ray source of voltage 80 kvp and a 90 mA beam current were used. A 3-dimensional data volume was reconstructed at 17 μm with use of the Feldkamp algorithm for cone beam CT geometry software. Images were exported into Amira (Mercury Computer Systems, Chelmsford, Massachusetts) as series of axial slices and were volume-rendered by setting the threshold slightly higher than the Hounsfield units of the Microfil (Flowtech) to display the vasculature.
After micro-CT imaging, the femoral arteries were cut into sequential 2-mm cross sections throughout the length of the occluded segment. In some instances, vessels were sectioned longitudinally. Sections were routinely stained with hematoxylin and eosin and Movat stains. In >80% of CTOs, ≥3 cross sections within the CTO were analyzable. The proximal and distal CTO segments were defined as the sections within the CTO that were immediately adjacent to the patent artery at the entry and exit site, respectively. All other sections were defined as the body of the CTO and measurements within this segment were averaged.
Morphometric measurements were performed with the Image J software (National Institutes of Health, Bethesda, Maryland). Lumen, media, and vessel cross-sectional area (CSA) were measured in Movat-stained sections. Integrity of the internal elastic lamina (IEL) was quantified by calculating the percentage of disrupted IEL (disrupted IEL circumference/overall IEL circumference × 100). Movat-stained sections also were used to calculate relative percent areas of 3 components of the CTO (lipid, microvessels, and neointimal tissue) at the 4 time points.
Inflammation was graded separately by 2 observers (R.J., J.B.) for the intima and media on a scale of 1 to 4 by counting the number of white blood cells within a high-power field (hpf) (×200 magnification): 0/hpf, 1 to 20/hpf, 21 to 50/hpf, and >50/hpf. Analysis of collagen content was performed with picrosirius red (PSR) staining. Qualitative collagen analysis was performed on PSR-stained slides by orientation-independent polarized microscopy (LC-PolScope, CRI Inc., Woburn, Massachusetts) (14). Quantitative analysis of collagen content was performed by morphometric measurement of the percentage of lumen area stained with PSR (15). Proteoglycan content was analyzed qualitatively in Alcian blue-stained cross sections.
The CTO microvessels within the occluded lumen were identified on Movat-stained sections as vascular structures lined with endothelial cells and with the occasional presence of medial smooth muscle cells (SMCs). These structures were often filled with Microfil. In some cases, red blood cells also were evident. Endothelial cell identification was confirmed with CD31 immunohistochemistry on selected slides. In each cross section, the number of microvessels, the CSA of each individual microvessel, and the overall microvessel (intima + media) CSA within the cross section were measured morphometrically with the use of Image J software (National Institutes of Health). For the identification of luminal cell types, immunohistochemistry was performed with the use of specific antibodies directed against macrophages (RAM 11, Dako, Mississauga, Ontario, Canada) and SMCs (alpha-SMC actin and desmin, Clone DE-U-10, Sigma, St. Louis, Missouri).
Data were analyzed by analysis of variance with the use of the generalized linear model procedure (proc GLM) in SAS (version 8.2, SAS Institute, Cary, North Carolina). Values are presented as mean ± SEM. For the comparison of means at each time point, least squares means and SEM estimates were calculated by SAS proc GLM, with the use of the Tukey-Kramer adjustment of p values for multiple comparisons. A value of p < 0.05, adjusted for multiple comparisons, was considered statistically significant.
Arterial CSA, within the external elastic lamina, rapidly decreased from 1.146 ± 0.096 mm2at 2 weeks to 0.327 ± 0.026 mm2at 6 weeks (p < 0.0001), with further (nonsignificant) reduction to 0.223 ± 0.012 mm2at 18 to 24 weeks (Figs. 1 and 2).⇓Lumen CSA, within the IEL, concomitantly decreased from 0.310 ± 0.014 mm2at 2 weeks to 0.108 ± 0.005 mm2at 18 to 24 weeks (p < 0.0001). A progressive decrease of IEL integrity also was observed, with missing IEL increasing from 4.9 ± 2.1% at 2 weeks to 16.2 ± 2.8% at 18 to 24 weeks.
Inflammatory, thrombotic, and lipid changes
An acute inflammatory infiltrate containing neutrophils and mononuclear cells peaked at 2 weeks with an inflammatory score at 3.5 ± 0.1 U, with progressive reductions at 6 weeks (2.7 ± 0.2 U, p = 0.0007) and 18 to 24 weeks (2.1 ± 0.1 U, p < 0.0001) (Fig. 1). There was a prominent macrophage infiltration within the lumen at 2 weeks, which was mixed with thrombotic material in the lumen (Fig. 3).By 6 weeks, macrophages were still evident within the occluded lumen, which no longer contained thrombus, and these macrophages frequently were situated adjacent to microvessels. At later time points, only rare macrophages were present within the lumen. Mean luminal macrophage number decreased from 2 weeks (280 ± 44 cells) to 6 weeks (60 ± 16 cells) and 12 weeks (18 ± 7 cells) (p < 0.002). At later time points there was an accumulation of extracellular lipid with an increase in the relative luminal CSA of lipids from 2 weeks to the 18- to 24-week time period (0 ± 0% to 20.7 ± 3.4%, p < 0.0001) (Fig. 1). At all time points, alpha-SMC actin-positive cells were present in the media of the vessel (Fig. 4).However, alpha-SMC actin–positive cells were only evident in the lumen of 2-week-old occlusions. These cells also were desmin negative, which led us to identify the cell type as a myofibroblast (16). At all later time points, the lumen cells were alpha-SMC actin–negative, except in the media of luminal microvessels.
At the 2-week time point, the formation of a proteoglycan-enriched extracellular matrix within the thrombotic occlusion was observed (Fig. 5).This matrix deposition occurred in a patchy pattern and was first evident in the middle of 2-week-old occlusions and then was present at both the proximal and distal ends of the CTO. The proteoglycan content progressively decreased beyond the early time points (2 and 6 weeks), with minimal or absent Alcian-blue staining at later time points. In contrast, lumen collagen content progressively increased over time.
Collagen was not evident in 2-week old CTO. At 6 weeks, the relative lumen collagen content was 10.7 ± 1.2% and increased to 15.0 ± 1.1% at 18 to 24 weeks. Polscope microscopy demonstrated the increased collagen density within the advanced CTO. Longitudinal sections of CTO at the ≥12-week time period demonstrated a region of densely packed collagen at the entrance to the CTO, “the proximal fibrous cap” (Fig. 6).
Neovascularization and CTO RBV
Both structural and functional analyses of CTO neovascularization were performed. Microvessels were already apparent within the intima and media as early as the 2-week time point. Total microvessel CSA was 3-fold greater in the occluded lumen than in the media at all time points. Total microvessel CSA increased 2-fold within the occlusion from 2 to 6 weeks (0.014 ± 0.002 mm2to 0.023 ± 0.005 mm2, p = 0.0008) together with a 3-fold increase in the size of individual intraluminal microvessels (0.0016 ± 0.0001 mm2to 0.0049 ± 0.0007 mm2, p < 0.0001) (Fig. 7).These structural changes were accompanied by a 3-fold increase in CTO RBV by MRI (5.1 ± 1.9% to 16.9 ± 2.7%, p = 0.0008) (Fig. 8).
At later time points (both 12 and 18 to 24 weeks), there was a significant reduction in intraluminal CTO neovascularization. At 18 to 24 weeks, there was a 79% reduction in RBV relative to 6 weeks (3.5 ± 1.1%, p < 0.0001) (Fig. 8), accompanied by a marked decrease in total microvessel CSA (0.017 ± 0.002 mm2, p = 0.011) (Fig. 7). There was a significant correlation between RBV and total microvessel CSA (r = 0.40, p < 0.03).
Micro-CT imaging demonstrated temporal and spatial differences in the vascular geometry. At the 2-week time point, the entrance appeared blunt, with a minimal amount of poorly defined channels extending into the soft thrombus (Fig. 9).At this time point, extravascular collaterals are already present. At 6 weeks, intraluminal recanalization channels with a “corkscrew-like” appearance were present at the proximal part of the occlusion. The endoluminal channels within the proximal portion were on the order of 50 μm in diameter. The midsection typically illustrated little contrast perfusion, with channels appearing discontinuous, making tracking of the vessels more difficult. At the late time point, there was regression of the proximal recanalization channel, and the entrance appeared blunt. In addition, scans also showed several small, highly fragmented channels appearing within the lumen, suggesting an apparent loss of continuity of the microvessels.
In this study, we identified 3 stages of CTO maturation: early (2 weeks), intermediate (6 to 12 weeks), and advanced (18 to 24 weeks). The early stage was characterized by an acute inflammatory response to vessel injury and initial patchy formation of extracellular matrix and myofibroblast infiltration within the thrombotic occlusion. This immature extracellular matrix was characterized by abundant proteoglycan content and low collagen density. During the initial part of the intermediate stage (6 weeks), the CTO had undergone marked negative arterial remodeling, IEL disruption, and intraluminal neovascularization, accompanied by increased CTO perfusion. During the latter intermediate stage (12 weeks), microvessels and relative blood volume within the CTO were already decreasing, which persisted into the advanced stage. The decreased perfusion in advanced CTO time points coincided with an increase in luminal collagen density and proteoglycan depletion.
The maturation of CTO represents a form of wound healing that is characterized by replacement of an initially proteoglycan-rich extracellular matrix into a collagen-rich scar at later time points. This pattern of healing is analogous to similar responses occurring after skin damage or various forms of arterial wall injury. However, the robust intraluminal neovascularization response seems unique to occluded vessels (both veins and arteries). Bidirectional thrombus formation is considered an essential component of the initiating event in the pathogenesis of human CTO development (1). Our experimental model specifically addresses the organization of thrombus within the arterial occlusion as a critical determinant of CTO maturation. Previous studies of thrombus organization have been almost exclusively limited to veins (17).
Initially, the freshly-formed thrombus contains platelets and erythrocytes within a fibrin mesh, which is followed by invasion of acute inflammatory cells (18). Endothelial cells also invade the fibrin lattice and form tube-like structures and microvessels within the organizing thrombi (17,19). There are few published data on the process of microvessel formation in arterial thrombi, which may differ from the process of venous neovascularization. In one study, it was reported that arterial thrombi recanalize less frequently and to a lesser extent than venous thrombi. The behavior of venous cells may also differ substantially from their arterial counterparts (20,21).
Inflammation is known as a potent trigger of neovascularization (22,23) and appeared to play an important role in our model. The neovascularization of CTO was preceded by an acute inflammatory response with prominent macrophage infiltration, particularly in perivascular regions. Previous studies have similarly shown high concentrations of macrophages in regions of recanalization in spontaneous human thrombi and in experimental animal arterial thrombi (24,25). Srivatsa et al. (6) likewise reported that microvessel content in human coronary CTOs correlated with degree of inflammation.
Our study provides several factors in support of a biologic basis for the clinical observation of increased guidewire failure to cross older CTOs. The physical properties identified by our study include marked negative remodeling that resulted in approximately 80% reduction in the overall size of the vessel by 6 weeks. This could potentially increase the difficulty of maintaining an intraluminal position of the guidewire. Moreover, for the first time, we have shown anatomic evidence of a “proximal fibrous cap,” a thickened structure at the entrance of the CTO containing particularly densely-packed collagen. Although collagen is present throughout the CTO, its prominence within the proximal fibrous cap likely acts as a distinct physical barrier to access the CTO.
The biological composition of a CTO also changes over time. Occlusions that were 2 weeks of age were characterized by a proteoglycan-rich matrix that is relatively soft and easy to traverse. At the later time points, proteoglycans had been replaced by collagen, which has considerably different biomechanical properties that resist guidewire penetration. Prominent temporal changes in CTO perfusion also were evident. Despite the absence of flow by angiography (because of its limited resolution of approximately 200 microns), contrast-MRI showed significant differences in RBVs, a surrogate for perfusion, at the various time points. There were transient and marked increases in microvessel CSA and RBV from 2 to 6 weeks, followed by reductions to low levels at the 2 later time points.
These findings were supported by the micro-CT images, which showed a prominent tapering “corkscrew-like” vascular structure in the proximal part of the CTO. The description of a tapering entrance of CTOs at coronary angiography has been correlated with successful crossing (7). However, this vascular structure regressed and was not visible at later time points. In addition, there also was fragmentation of intraluminal microvessels evident at the later time points. The discontinuity of the microvascular network and reduced CTO perfusion represent an unfavorable topography for guidewire crossing, particularly with the increased rigid collagen-rich composition in the intervening extracellular matrix at these later time points.
Our finding of a prominent microvessel network within the CTO, despite the absence of perfusion by angiography, is in agreement with human pathological studies (6,7). Intraluminal neovascularization of the CTO may facilitate successful guidewire crossing (6–8). On the basis of our measurements of individual microvessel CSA, it is doubtful that conventional guidewires (360-micron diameter) actually use an individual microvessel for crossing. However, it is possible that groups of microvessels in close proximity to each other or altered tissue compliance characteristics in a highly perfused segment may facilitate guidewire crossing. The ability to detect the presence of microvessels and enhance their formation may underlie future diagnostic and therapeutic strategies.
Relevance to human CTOs
Although CTOs are common and difficult to treat with percutaneous coronary intervention (PCI), data from human pathological studies are sparse and anecdotal. A relevant animal model is necessary for systematic analysis of the process of CTO maturation and development of innovative therapeutic approaches. Our experimental model does have limitations because it lacks an atherosclerotic substrate and calcification that may potentially impact the CTO maturation process. However, it recreates many features that have been recognized in the natural history of CTO (1), including an initial thrombotic occlusion, acute inflammatory response, negative arterial remodeling, and the presence of microvessels and collagen-rich fibrous tissue. Our study is the first to systematically document the temporal changes during the process of CTO maturation that may underlie the difficulty in performing PCI in these lesions. Importantly, this study also correlated findings from noninvasive imaging with histological data, thus laying the groundwork for future studies to examine the predictive value of imaging in selecting patients for PCI in addition to more complex atherosclerotic animal models.
The pathology and interventional success rates of CTO are quite different at various time points. Our findings of decreased vascular perfusion and intraluminal neovascularization in concert with collagen accretion at later time points that are typically characterized by very low success rates have important clinical relevance. First, they offer rational targets for improving success rates, such as altering collagen content by enzymatic digestion (9, 26) and/or enhancing intraluminal microvascular network formation; second, our study supports development of noninvasive imaging modalities to improve tissue characterization and identification of favorable and unfavorable lesion characteristics as part of pre-procedure revascularization planning. The significance of CTO blood volume measurements as a predictor of CTO crossing requires validation in human coronary CTO studies. Plans are underway to noninvasively image human CTO for blood volume measurements with the use of MR contrast reagents before attempts at revascularization are made. Further study is required to determine whether transformation of advanced CTO structural characteristics to those of an immature occlusion, by induction of microvessel formation and/or degradation of collagen content within the extracellular matrix, will enhance clinical CTO percutaneous revascularization success rates.
Dr. Jaffe is a Research Fellow of the Heart and Stroke Foundation of Canada. This study was funded by the Canadian Institute of Health Research (MOP-53325, CTP-82943, and MOP-12492).
- Abbreviations and Acronyms
- cross-sectional area
- computed tomography
- chronic total arterial occlusion
- high power field
- internal elastic lamina
- magnetic resonance imaging
- percutaneous coronary intervention
- picrosirius red
- relative blood volume
- Received February 21, 2008.
- Revision received August 26, 2008.
- Accepted September 4, 2008.
- American College of Cardiology Foundation
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