Lumen loss in transplant coronary artery disease is a biphasic process involving early intimal thickening and late constrictive remodeling: results from a 5-year serial intravascular ultrasound study.

BACKGROUND: Coronary artery disease is the major cause of late cardiac allograft failure. However, few data exist regarding the natural history of changes in intimal and external elastic membrane (EEM) areas after heart transplantation. METHODS AND RESULTS: In 38 transplant recipients, serial intravascular ultrasound examinations were performed 3.7+/-2.2 weeks after transplantation and annually thereafter for 5 years. In 59 coronary arteries, we compared 135 matched segments among serial studies. In each segment, intravascular ultrasound images were digitized at 1-mm intervals, and mean values of EEM and lumen and intimal areas were analyzed. In the first year after transplantation, the intimal area increased significantly from 1.8+/-1.6 to 3.0+/-2.1 mm(2) (P<0.001). Subsequently, the annual increase in intimal area decreased. EEM area did not change during the first year; however, between years 1 and 3, significant expansion of EEM area occurred (15.4+/-4.6 to 17.2+/-5.4 mm(2), P<0.001). Thereafter, EEM area decreased significantly from 17.2+/-5.4 mm(2) (year 3) to 15.1+/-4.9 mm(2) (year 5, P=0.01). Different mechanisms of lumen loss were observed during 2 phases after transplantation: early lumen loss primarily caused by intimal thickening and late lumen loss caused by EEM area constriction. CONCLUSIONS: This serial ultrasound study revealed that most of the intimal thickening occurred during the first year after heart transplantation. Changes in the EEM area showed a biphasic response, consisting of early expansion and late constriction. Thus, different mechanisms of lumen loss were observed during the early and late phases after transplantation.

Arterial remodeling and coronary artery disease: the concept of "dilated" versus "obstructive" coronary atherosclerosis.

Traditionally, the development of coronary artery disease (CAD) was described as a gradual growth of plaques within the intima of the vessel. The outer boundaries of the intima, the media and the external elastic membrane (EEM), were thought to be fixed in size. In this model plaque growth would always lead to luminal narrowing and the number and severity of angiographic stenoses would reflect the extent of coronary disease. However, histologic studies demonstrated that certain plaques do not reduce luminal size, presumably because of expansion of the media and EEM during atheroma development. This phenomenon of "arterial remodeling" was confirmed in necropsy specimens of human coronary arteries. More recently, the development of contemporary imaging technology, particularly intravascular ultrasound, has allowed the study of arterial remodeling in vivo. These new imaging modalities have confirmed that plaque progression and regression are not closely related to luminal size. In this review, we will analyze the role of remodeling in the progression and regression of native CAD, as well as its impact on restenosis after coronary intervention.

Visualization of the spatial distribution of silver in histological sections.

The distribution of silver surrounding silver-containing implant devices can be clearly demonstrated using an autometallographic technique originally described by Danscher.

Quantitative assessment of the tissue response to implanted biomaterials.

The tissue response to a small number of polymeric biomaterials was studied using monoclonal antibodies specific for certain inflammatory cell types, to develop a reliable and accurate method for the quantitative evaluation of biocompatibility. The sites of antibody binding were identified using an avidin-biotin staining procedure and the sections evaluated using a computer-aided image analysis system. The staining technique successfully demonstrated both polymorphonuclear leucocytes and macrophages in tissue samples containing polymeric biomaterials. The image analysis system facilitated the measurement of up to 30 cell-related parameters and allowed a large number of cells to be analysed.

Quantitative analysis of inflammatory cells in aortic atherosclerosis of young adults.

Cellular analysis of aortic atherosclerotic lesions has been pursued extensively in recent years, although most of these investigations have involved the detection of inflammatory cells in chronically diseased tissue or artificially induced atherosclerosis in an animal model. Few studies have attempted to quantify accurately, using computer analysis systems, the degree of cellular infiltration in a statistically significant number of samples, in tissue from young adults. In this study, segments of human aortae were collected at autopsy from 29 individuals ranging in age from 15 to 35 years. The tissue was embedded in paraffin and stained using routine histological and automated immunohistochemical staining techniques. The sections were evaluated using advanced image analysis techniques to investigate the differences in cellular composition and cell activation between the dorsal and ventral aspects of the human aorta and to correlate these findings to the age of the subjects. These regions have been previously shown to have a high (dorsal) and low (ventral) probability of developing sudanophilic lesions. Our data demonstrated that statistically different cell populations exist in the dorsal and ventral regions of each vessel. The dorsal aspect (i.e., high-probability region) had a greater number of HAM56(+) (36.9% increase,p = 0.0002) and HLA-DRα(+) cells (44.2% increase,p = 0.0035) than did the ventral surface (i.e., low-probability region), although there were no significant differences in the number of CD43(+) lymphocytes. When grouped according to age, results showed significant increases in the dorsal region when considering HAM56(+) and HLA-DRα(+) cells (p = 0033 and 0.046, respectively). Morphologically, a greater number of foam cell aggregates were found to occur in the dorsal region of the vessel than in the ventral portion. Our results indicate that the microarchitecture and cellular composition of the dorsal and ventral aorta are significantly different, with these variations becoming more marked with age.

Evaluation of three-dimensional segmentation algorithms for the identification of luminal and medial-adventitial borders in intravascular ultrasound images.

Intravascular ultrasound (IVUS) provides direct depiction of coronary artery anatomy, including plaque and vessel area, which is important in quantitative studies on the progression or regression of coronary artery disease. Traditionally, these studies have relied on manual evaluation, which is laborious, time consuming, and subject to large interobserver and intraobserver variability. A new technique, called active surface segmentation, alleviates these limitations and makes strides toward routine analyses. However, for three-dimensional (3-D) plaque assessment or 3-D reconstruction to become a clinical reality, methods must be developed which can analyze many images quickly. Presented is a comparison between two active surface techniques for three-dimensional segmentation of luminal and medial-adventitial borders. The force-acceleration technique and the neighborhood-search technique accurately detected both borders in vivo (r2 = 0.95 and 0.99, Williams' index = 0.67 and 0.65, and r2 = 0.95 and 0.99, WI = 0.67 and 0.70, respectively). However, the neighborhood-search technique was significantly faster and required less computation. Volume calculations for both techniques (r2 = 0.99 and r2 = 0.99) also agreed with a known-volume phantom. Active surface segmentation allows 3-D assessment of coronary morphology and further developments with this technology will provide clinical analysis tools.

Assessing spectral algorithms to predict atherosclerotic plaque composition with normalized and raw intravascular ultrasound data.

Spectral analysis of backscattered intravascular ultrasound (IVUS) data has demonstrated the ability to characterize plaque. We compared the ability of spectral parameters (e.g., slope, midband fit and y-intercept), computed via classic Fourier transform (CPSD), Welch power spectrum (WPSD) and autoregressive (MPSD) models, to classify plaque composition. Data were collected ex vivo from 32 human left anterior descending coronary arteries. Regions-of-interest (ROIs), selected from histology, comprised 64 collagen-rich, 24 fibrolipidic, 23 calcified and 37 calcified-necrotic regions. A novel quantitative method was used to correlate IVUS data with corresponding histologic sections. Periodograms of IVUS samples, identified for each ROI, were used to calculate spectral parameters. Statistical classification trees (CT) were computed with 75% of the data for plaque characterization. The remaining data were used to assess the accuracy of the CTs. The overall accuracies for normalized spectra with CPSD, WPSD and MPSD were, respectively, 84.7%, 85.6% and 81.1% (training data) and 54.1%, 64.9% and 37.8% (test data). These numbers were improved to 89.2%, 91.9% and 89.2% (training) and 62.2%, 73% and 59.5% (test) when the calcified and calcified-necrotic regions were combined for analysis. Most CTs misclassified a few fibrolipidic regions as collagen, which is histologically acceptable, and the unnormalized and normalized spectra results were similar.

Comparison of texture analysis methods for the characterization of coronary plaques in intravascular ultrasound images.

Intravascular Ultrasound (IVUS) is a diagnostic imaging technique that provides tomographic visualization of coronary arteries. The aim of this study was to evaluate five texture analysis techniques and determine their ability to distinguish between plaque lesions of different composition. Using histological correlation, regions of calcified, fibrous, and necrotic core plaque were chosen from 27 coronary plaques. First-order statistics, Haralick's method, Laws' texture energy method, the neighborhood gray-tone difference matrix method, and texture spectrum features were examined using discriminant analysis. Self-validation indicated that Haralick's method yielded the most accurate results, with resubstitution and cross-validation error rates of 0.00 and 14.76%, respectively. Further optimization gave error rates of 6.67%, using only two discriminating features, IDM and entropy.

Three-dimensional segmentation of luminal and adventitial borders in serial intravascular ultrasound images.

Intravascular ultrasound (IVUS) provides exact anatomy of arteries, allowing accurate quantitative analysis. Automated segmentation of IVUS images is a prerequisite for routine quantitative analyses. We present a new three-dimensional (3D) segmentation technique, called active surface segmentation, which detects luminal and adventitial borders in IVUS pullback examinations of coronary arteries. The technique was validated against expert tracings by computing correlation coefficients (range 0.83-0.97) and William's index values (range 0.37-0.66). The technique was statistically accurate, robust to image artifacts, and capable of segmenting a large number of images rapidly. Active surface segmentation enabled geometrically accurate 3D reconstruction and visualization of coronary arteries and volumetric measurements.

The clinical morphology of human atherosclerotic lesions. Lessons from the PDAY Study. Pathobiological Determinants of Atherosclerosis in Youth.

The PDAY study addresses the role traditional risk factors play in the development and progression of arterial lesions in youth (15 to 34 years of age). This autopsy based study indicates that in the aorta and in the right coronary artery, atherosclerosis is positively associated with cholesterol (LDL+VLDL), glucose intolerance, smoking, hypertension, and obesity and negatively associated with HDL-cholesterol. When a high cholesterol group (> 240 mg/dl) is compared to a low cholesterol group (< 200 mg/dl) early fatty sudanophilic lesions decrease in the aorta from 35% to 29% (p < 0.03) surface area, while raised atherosclerotic lesions are lowered (from 5%) in the high cholesterol group (to 3%) in the low cholesterol group; p < 0.02). Clearly, risk factors particularly cholesterol levels, directly affect the development of atherosclerosis in the coronary arteries and the aorta of young people. The data presented suggest that risk factor modification would be beneficial in young people as from 15 years of age.

Image analysis in the evaluation of biomaterials.

An examination regime, based on a computer-aided image analysis system, has been developed for the quantitative evaluation of the local tissue response to biomaterials. This procedure involves the immunoenzymic staining of tissue sections using monoclonal antibodies specific for certain inflammatory cell types. An avidin-biotin-horseradish peroxidase staining method is used to identify antibody binding sites and the sections are assayed using a computer-aided image analysis system. This regime facilitates the rapid and accurate measurement of 30 cell related parameters in sections stained for macrophages, polymorphonuclear leucocytes, and other cells.

Automated morphometry of coronary arteries with digital image analysis of intravascular ultrasound.

We designed and tested digital image processing strategies to perform fully automated segmentation of luminal and medial-adventitial boundaries in intravascular ultrasound images of human coronary arteries. Automated segmentation is an essential tool for advanced techniques of clinical visualization and quantitative measurement. Vascular compliance measurements and three-dimensional reconstructions are demonstrated as examples of such applications. Digital image processing was performed in three phases: (1) preprocessing, including a polar transform, local contrast enhancement, and speckle noise filtering; (2) segmentation, involving radial scanning, region growing, or cost-function minimization techniques; and (3) postprocessing, involving dropout filtering and outline smoothing. Cross-sectional areas were compared with manual tracings from experienced operators and showed good agreement. The algorithm bias ranged from -0.34 to 1.18 mm2; interclass and intraclass correlation coefficients ranged from 0.83 to 0.94. The designed techniques currently allow fully automated segmentation without operator interaction of the luminal and, if present, medial-adventitial boundary.

Automated identification of stained cells in tissue sections using digital image analysis.

OBJECTIVE: To develop a novel automated image analysis system to differentiate immunohistochemically stained cells from background. STUDY DESIGN: Cell segmentation was performed by applying global thresholding algorithms to find an approximate threshold at which cells could be separated from background followed by a novel refinement algorithm to erode edge pixels of the region. To separate overlapping cells, a new decomposition method was developed that uses both semantic knowledge and high-level relational information. Both the cell segmentation and separation methods were evaluated on images of stained tissue sections and the manually outlined cell areas and numbers compared to the computed. RESULTS: Macrophage areas computed at the first stage by Otsu's algorithm did not differ significantly (P = .07) from those traced manually, while the areas computed by Kittler's and Kurita's algorithms did not agree (P < .01). Both Otsu's and Kurita's algorithms performed well when combined with edge pixel erosion. Kittler's algorithm proved unsuccessful even with edge erosion. Comparison of the computed and manually determined cell numbers showed a significant correlation, and regression analysis resulted in the unity curve. CONCLUSION: A combination of global thresholding and a novel edge erosion technique allowed identification of immunohistochemically stained macrophages; the computed cell areas agreed with the manual results.

Three-dimensional reconstruction of the coronary artery wall by image fusion of intravascular ultrasound and bi-plane angiography.

BACKGROUND: Intravascular ultrasound (IVUS) is becoming increasingly accepted for assessing coronary anatomy. However, its utility in visualizing and quantifying coronary morphology has been limited by its 2D tomographic nature. This study presents a 3D reconstruction technique that accurately preserves 3D geometric information. METHODS AND RESULTS: Images obtained from manual IVUS pullbacks and continuous bi-plane angiography were fused, using angiography to reconstruct the transducer trajectory and aid in solving for the correct rotational orientation. A novel 3D active surface method automatically identified the luminal and medial-adventitial borders which, when superimposed on the transducer trajectory, could be surface-rendered for visualization and morphometry. Segmentation agreed well with manual assessment, and 3D luminal shape matched that of angiography when projected to 2D. CONCLUSIONS: We conclude that this method provides an accurate reconstruction of the vessel's anatomy, which accounts for the true curvature of the vessel.

Quantitative comparison of immunohistochemical staining intensity in tissues fixed in formalin and Histochoice.

Formaldehyde fixatives have traditionally been used to preserve tissues as they impart excellent morphological preservation. Formaldehyde fixes tissue by cross linking, a process which can reduce the antigenicity of tissue and weakens the intensity of immunohistochemical stains. Preliminary studies have shown that Histochoice tissue fixative offers equal or greater staining intensity than neutral buffered formalin (NBF). This study compares these fixatives quantitatively and presents the results in unambiguous statistical terms. Tissue samples were collected, bisected, and fixed in NBF or Histochoice. The sections were stained with a total of 21 antibodies, and color images were collected. The hue, saturation, and value were determined for each positive pixel and an ANOVA performed. Small differences in hue were noted in 8 of 21 cases. Histochoice-fixed tissue gave a greater mean saturation than NBF with 57.1% of the antibodies tested. No significant difference in the saturation was detected in 28.6% of the cases; NBF gave higher mean saturation levels with only 14.3% of the antibodies. Histochoice-fixed tissue was found to give lower values in 66.7% of cases than those prepared with NBF, indicating darker staining. These results show that Histochoice produces staining intensity that is comparable, and in many cases superior, to formalin.

Assessment of coronary compensatory enlargement by three-dimensional intravascular ultrasound.

Several techniques have been used to demonstrate that human arteries respond to atherosclerosis by increasing their total arterial area to prevent a decrease in blood flow. Three-dimensional reconstructions of coronary arteries can document this compensatory response accurately and specifically. Seven human coronary arteries were reconstructed using intravascular ultrasound and biplane angiography, and vessel geometries were quantified. In all seven vessels, as plaque area increased, overall vessel area increased (R = 0.986, 0.933, 0.984, 0.678, 0.763, 0.963, and 0.830), but luminal cross-sectional area did not significantly decrease. Focal compensatory enlargement was identified in each vessel, and in some cases this response appeared to occur until the vessel was 65% occluded. Luminal enlargement near the proximal ends was attributed to the natural taper of the vessel. The semi-automated, three-dimensional segmentation technique used in this study allows reproducible quantification, as there is no subjective manual tracing involved. Following the intravascular ultrasound transducer in time and space with biplane angiography allows for accurate reconstruction with or without automated pullback devices. Information on the rate of change of vessel measurements is also presented, which, when combined with visualization of accurate 3D geometry, provides a unique assessment of coronary compensatory enlargement. This reconstruction technique can be applied in a clinical environment with no major modification.

Reduction in myocardial infarct size by basic fibroblast growth factor after temporary coronary occlusion in a canine model.

BACKGROUND: Basic fibroblast growth factor (bFGF) has been shown to reduce infarct size in canine acute myocardial infarction; however, the mechanism of tissue salvage remains uncertain. We evaluated the effect of bFGF on infarct size in a model of acute infarction in which coronary occlusion was followed by prolonged reperfusion and sought to determine whether reperfusion attenuates the stimulus for myocardial neovascularization. METHODS AND RESULTS: Anesthetized dogs undergoing 4-hour balloon occlusion of the left anterior descending coronary artery were treated with intracoronary bFGF (n = 8) or vehicle (n = 6). Ten-microgram doses of bFGF were administered 10 minutes after occlusion and again immediately before reperfusion. Left ventriculograms were obtained before occlusion, after reperfusion, and preceding euthanasia on day 7. Infarct size, expressed as a percentage of the area at risk, was reduced in bFGF-treated dogs (13.7 +/- 2.1% versus 28 +/- 3.4%; P = .002). Changes in left ventricular ejection fraction, capillary density, and cellular proliferation-assessed immunohistochemically with factor VIII and proliferating cell nuclear antigen antibodies-were similar in both groups. To assess coronary vasomotor responses to bFGF, a separate hemodynamic study was performed in five anesthetized nonischemic dogs in which incremental bFGF doses up to 100 micrograms induced no vasodilator response. CONCLUSIONS: Treatment with bFGF was associated with a reduction in infarct size without hemodynamic effects or evidence of neovascularization. These data suggest that bFGF mediates myocardial salvage independently of angiogenesis and that reperfusion after infarction may attenuate the stimulus for neovascularization.

Quantification of intramural calcification in coronary intravascular ultrasound images with automated image analysis.

BACKGROUND: Recent studies have documented the utility of intravascular ultrasonography in quantifying coronary morphologic characteristics and determining an appropriate intervention. Unfortunately, its potential for quantifying lesion calcification is limited by subjective evaluation and manual tracing. The aim of this study was to develop an objective automated method for quantifying calcification in intracoronary images with digital image analysis. METHODS: Images of human coronary arteries acquired with a 30 MHz intracoronary ultrasound catheter were evaluated with digital image analysis and compared with manual tracings. Calcifications were automatically identified as highly echogenic regions detected by global thresholding within sectors of acoustic shadowing defined as regions devoid of texture. RESULTS: The mean percentage agreement, sensitivity, and specificity of detecting calcification in 1-degree sectors of calcified vessels were 82%, 73%, and 87%, respectively. Similar results were obtained in noncalcified images. CONCLUSION: The accuracy of this automated technique was comparable to interoperator and intraoperator variability in manually tracing calcification.