Willem Jacob

Apoptosis and Related Proteins in Different Stages of Human Atherosclerotic Plaques

BACKGROUND The transition of a fatty streak into an atherosclerotic plaque is characterized by the appearance of focal and diffuse regions of cell death. We have investigated the distribution of apoptotic cell death and apoptosis-related proteins in early and advanced atherosclerotic lesions. METHODS AND RESULTS Human atherosclerotic plaques were studied by whole-mount carotid endarterectomy specimens (n=18). This approach allowed comparison of adaptive intimal thickenings, fatty streaks, and advanced atherosclerotic plaques of the same patient. The fatty streaks differed from adaptive intimal thickenings by the presence of BAX (P<0.01), a proapoptotic protein of the BCL-2 family. Both regions were composed mainly of smooth muscle cells (SMCs), and macrophage infiltration was low and not different. Apoptosis, as detected by DNA in situ end labeling (terminal deoxynucleotidyl transferase end labeling [TUNEL] and in situ nick translation) was not present in these regions. Apoptosis of SMCs and macrophages, however, was present in advanced atherosclerotic plaques that were present mainly in the carotid sinus. A dense infiltration of macrophages (5.8+/-3% surface area) was present in these advanced atherosclerotic plaques. Cytoplasmic remnants of apoptotic SMCs, enclosed by a cage of thickened basal lamina, were TUNEL negative and remained present in the plaques as matrix vesicles. CONCLUSIONS We conclude that SMCs within human fatty streaks express BAX, which increases the susceptibility of these cells to undergo apoptosis. The localization of these susceptible SMCs in the deep layer of the fatty streaks could be important in our understanding of the transition of fatty streaks into atherosclerotic plaques, which are characterized by regions of cell death. Matrix vesicles are BAX-immunoreactive cytoplasmic remnants of fragmented SMCs that can calcify and may be considered the graves of SMCs that have died in the plaques.

Triphasic sequence of neointimal formation in the cuffed carotid artery of the rabbit.

A nonocclusive silicone cuff placed around the rabbit carotid artery results in a diffuse intimal thickening. The early stages of this phenomenon were studied by light microscopy, immunohistochemistry, and electron microscopy. Neointimal formation appeared to be triphasic. The first phase started 2 hours after cuff placement, with vascular infiltration by polymorphonuclear leukocytes (PMNs). In the second phase, starting within 12 hours, 1.90 +/- 0.36% of the medial smooth muscle cells (SMCs) were replicating, as demonstrated by their immunoreactivity for proliferating cell nuclear antigen (PCNA). The third phase was characterized by the appearance, from day 3 onward, of subendothelial SMCs that were immunoreactive for alpha-SMC actin and vimentin. A few cells showed immunoreactivity for PCNA. During this phase all the PMNs disappeared, but SMC replication in the media was still present, as indicated by the presence of mitoses and the persisting immunoreactivity for PCNA (0.76 +/- 0.22% at day 7). In the third phase the number of subendothelial cells increased (104 +/- 15 SMC nuclei per section at day 7, of which 8.89 +/- 2.26% were PCNA-positive) and was associated with deposition of collagen type IV and fibronectin. At 14 days a complete, circular neointima was present and contained 2.13 +/- 0.28% replicating SMCs. The media showed 0.44 +/- 0.08% cell-cycling SMCs, which was still four times higher than normal. During the first week there was also a significantly higher PCNA activity in the media of sham-operated carotid arteries (no cuff present) than in nonsurgical ones. However, this did not lead to the formation of a neointima. We conclude that in the cuff system SMC replication in the media precedes the neointimal formation. The system can be used to study SMC replication, migration, and neointimal formation with minimal medial SMC damage.

Luminal Foam Cell Accumulation Is Associated With Smooth Muscle Cell Death in the Intimal Thickening of Human Saphenous Vein Grafts

BACKGROUND Occlusion of saphenous vein grafts is a major problem after coronary artery bypass graft surgery. Diffuse intimal thickening develops in all implanted aortocoronary saphenous vein grafts within 6 months to 1 year. In some regions of the thickened intima, foam cells accumulate along the luminal margin. This particular morphology resembles the morphology of unstable atherosclerotic plaques as they occur in coronary arteries. In the present study, we focused on the possible topographic relation between luminal foam cell accumulation and cell death of smooth muscle cells (SMCs) within the adjacent thickened intima. METHODS AND RESULTS Segments of occluded and suboccluded implanted human aortocoronary saphenous vein grafts were obtained during reintervention coronary artery bypass graft surgery in 30 patients. In the regions of the vein grafts with luminal foam cell accumulation, the percentage of SMC alpha-actin immunoreactive area of the superficial intimal thickening was 6 +/- 1.4%, which was different from the 17.6 +/- 2.3% of the deep intimal thickening. A strong negative correlation between the number of foam cell nuclei and the percentage of SMC alpha-actin immunoreactive area in the adjacent superficial intimal thickening was present (r = -.77, P < .001). Within the superficial intimal thickening, cytoplasmic and DNA fragmentation could be detected, which points to apoptotic cell death. A fraction of the cytoplasmic fragments fitted the ultrastructural characteristics of matrix vesicles and showed pronounced calcium and phosphorus accumulation as demonstrated with the use of x-ray microanalysis. CONCLUSIONS The close spatial relation among foam cell accumulation, pronounced intimal SMC loss, and cell death suggests the presence of a foam cell-derived factor that can induce cell death in the SMC population of the intimal thickening. The depletion of the intimal SMC population could promote plaque rupture and thrombotic complications in the grafts.

The endothelium during cuff-induced neointima formation in the rabbit carotid artery.

Intimal thickening in human arteries is considered as a site of predilection for atherosclerosis. The placement of a flexible, physically nonconstrictive, silicone cuff around the rabbit carotid artery induced a neointima composed of smooth muscle cells (SMCs) within 14 days. To investigate possible alterations of the endothelial cells (ECs) during neointima formation, their morphology was examined with scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal microscopy. In the early postoperative period (6 hours), both cuffed and sham-operated arteries demonstrated small foci (5 to 200 microns) of denudation, presumably as a consequence of the manipulation. Within 24 hours the luminal surface of the cuffed and sham-operated arteries was completely covered with endothelium, which remained continuous throughout the study. However, after 1 week the ECs of the cuffed arteries contained a pronounced rough endoplasmic reticulum. From 6 hours until 3 days, polymorphonuclear leukocytes infiltrated the cuffed but not the sham-operated arteries from the lumen. Subendothelial SMC accumulation in the cuffed arteries began after this time period. At day 14 a full-blown neointima composed of longitudinally oriented SMCs had formed in the cuffed arteries. The sham-operated arteries did not develop a neointima. During neointima formation immunoreactivity for von Willebrand factor (vWf) increased in the ECs, and vWf was deposited in the extracellular spaces of the neointima. At day 14 the area of vWf deposits correlated positively with the area of the neointima (r = .73, P < .001). In subsequent weeks, the intimal area did not increase, and vWf deposits vanished from the neointimal matrix. The endothelium of the sham-operated arteries showed no change in vWf immunoreactivity compared with untreated arteries throughout the study. The altered ultrastructural morphology of the ECs and the concurrent vWf deposition in cuffed but not in sham-operated arteries point to alterations in EC function during the development of the neointima. The vWf secretion could possibly lead to increased adhesiveness of the extracellular matrix for the ECs as well as modulate neointima formation.

Automated breast tumor diagnosis and grading based on wavelet chromatin texture description

In this paper, wavelets were employed for multi-scale image analysis to extract parameters for the description of chromatin texture in the cytological diagnosis and grading of invasive breast cancer. Their value was estimated by comparing the performance of co-occurrence, densitometric, and morphometric parameters in an automated K-nearest neighbor (Knn) classification scheme based on light microscopic images of isolated nuclei of paraffin-embedded tissue. This design allowed a multifaceted cytological retrospective study of which the practical value can be judged easily. Results show that wavelets perform excellently with classification scores comparable with densitometric and co-occurrence features. Moreover, because wavelets showed a high additive value with the other textural groups, this panel allowed a very profound description with higher recognition scores than previously reported (76% for individual nuclei, 100% for cases). Morphometric parameters performed less well and only slightly increased correct classification. The major drawback, besides image segmentation errors demanding operator supervision, emanated to be the few false-negative cases, which restrict the immediate practical use. However, an enlargement of the parameter set may avoid this misclassification, resulting in an applicable expert system of practical use.

Computer-assisted differential diagnosis of malignant mesothelioma based on syntactic structure analysis.

BACKGROUND Malignant mesothelioma, a mesoderm-derived tumor, is related to asbestos exposure and remains a diagnostic challenge because none of the genetic or immunohistochemical markers have yet been proven to be specific. To assist in the identification of mesothelioma and to differentiate it from other common lesions at the same location, we have tested the performance of syntactic structure analysis (SSA) in an automated classification procedure. MATERIALS AND METHODS Light-microscopic images of tissue sections of malignant mesothelioma, hyperplastic mesothelium, and adenocarcinoma were analyzed using parameters selected from the Voronoi diagram, Gabriels graph, and the minimum spanning tree which were classified with a K-nearest-neighbor algorithm. RESULTS Results showed that mesotheliomas were diagnosed correctly in 74% of the cases; 76% of the adenocarcinomas were correctly graded, and 88% of the mesotheliomas were correctly typed. The performance of the parameters was dependent on the obtained classification (i.e., tumor-tumor versus tumor-benign). CONCLUSIONS Our results suggest that SSA is valuable in the differential classification of mesothelioma and that it supplements a visually appraised diagnosis. The recognition scores may be increased by a combination of SSA with, for example, cellular or nuclear parameters, measured at higher magnifications to form a solid base for fully automated expert systems.

Histological Study of the Thin Replacement Membrane of Human Tympanic Membrane Perforations

A histological study was done on the thin, nearly transparent replacement membrane of tympanic membrane perforations. Human tympanic membranes that were rejected for transplantation, were studied by light and electron microscopy. The abrupt reduction in thickness at the margin of the covered perforation, is entirely due to the reduction of the lamina propria. Even in the thinnest parts of the replacement membrane, a lamina propria is present, separated by continuous basement membranes from the epithelium and mucosa, and measuring no more than some 2-3 microns in thickness. This lamina propria consists of fibrils and interfibrillar matrix, but fibroblasts appear to be lacking. The epithelial layer does not contain basal cells, confirming the thesis that the upper layers are not generated by in situ proliferation, but that they have migrated from the periphery.

Data representation and reduction for chromatin texture in nuclei from premalignant prostatic, esophageal, and colonic lesions

BACKGROUND To identify nuclei and lesions with great specificity, a large set of karyometric features is arranged in the form of a linear profile, called a nuclear signature. The karyometric feature values are normalized as z-values. Their ordering along the profile axis is arbitrary but consistent. The profile of the nuclear signature is distinctive; it can be characterized by a new set of variables called contour features. A number of data reduction methods are introduced and their performance is compared with that of the karyometric features in the classification of prostatic, colonic, and esophageal lesions. METHODS Contour characteristics were reduced to descriptive statistics of the set of z-values in the nuclear signature and to sequence information. The contour features derived were (1) relative frequencies of occurrence of z-values and of their differences and (2) co-occurrence statistics, run lengths of z-values, and statistics of higher-order dependencies. Performance was evaluated by comparing classification scores of diagnostic groups. RESULTS Rates for correct classification by karyometric features alone and contour features alone indicate equivalent performance. Classification by a combined set of features led to an increase in correct classification. CONCLUSIONS Image analysis and subsequent data reduction of nuclear signatures of contour features is a novel method, providing quantitative information that may lead to an effective identification of nuclei and lesions.

Value of morphometry, texture analysis, densitometry, and histometry in the differential diagnosis and prognosis of malignant mesothelioma

Malignant mesothelioma is a tumour with increasing incidence due to widespread use of its causative agent, asbestos, in the past decades. The poor survival necessitates a correct differentiation from other lesions at the same site, such as hyperplastic mesothelium and carcinomas metastatic to pleura or peritoneum. Since genetic and immunohistochemical markers are not absolutely differentiating, the diagnosis is based on the histology complemented with (immuno)histochemistry. However, as the tumour presents itself in numerous heterogeneous histological forms, visual evaluation is extremely difficult. In order to evaluate the prognostic and diagnostic performance of syntactic structure analysis (SSA), chromatin texture analysis, densitometry, and morphometry, an automated KNN‐classification system has been used to compare Feulgen‐stained tissue sections of hyperplastic mesothelium, malignant mesothelioma, and pulmonary adenocarcinoma. In addition, we also studied most discriminative aspects in the differentiation, typing, and prediction of survival. The results indicate that for the diagnosis of malignant mesothelioma, chromatin texture parameters outperform SSA, densitometry, and morphometry (recognition score=96·8 per cent). Most discriminative parameters highlight spatial patterns of the chromatin distribution that are hard to appraise visually and directly show the benefits of a quantitative approach. Typing of the tumour is best described by SSA parameters, relating to the spatial arrangement of the cells in the tissue (recognition score=94·9 per cent). In survival time classifications, chromatin texture yields the highest recognition score (82·9 per cent), although accurate estimations are unreliable due to a large degree of misclassification. Copyright

Contact sites between the inner and outer mitochondrial membranes in stunned versus hibernating myocardium

This study characterizes the energy state and the influence of calcium on myocardial stunning and chronic hibernation via the quantification of a calcium-sensitive phenomenon known as mitochondrial contact sites. For stunning, the left anterior descending artery of mongrel dogs was occluded for 15 minutes, followed by a 150-minute reperfusion; for chronic hibernation, we used human biopsies obtained during coronary artery bypass graft (CABG) from viable (positron emission tomography-controlled) asynergic areas. Both sample groups were processed for electron microscopy, and the ratio of surface densities of contact sites to mitochondrial membranes was quantified with morphometry. Therefore a cycloidal pattern was superimposed on the electron micrographs, and the ratio between the total number of intersections between cycloids and contact sites and the total number of intersections between cycloids and mitochondrial membranes is the ratio of surface densities (Ss). In stunned cells, Ss = 0.46 ± 0.06, which is significantly higher than the ratio in the normokinetic cells, Ss = 0.355 ± 0.003, although the general ultrastructure of the subcellular compartments is practically identical to those in the normoxic area. The distinction between cells affected by chronic hibernation and normal cells was based on structural criteria. The ratio of surface densities, expressed Ss = 0.27 ± 0.05, was significantly lower than the ratio in the normoxic area (Ss = 0.356 ± 0.005). The high ratio of surface densities in the stunned cells lends credibility to the notion that stunning implies an increased intracellular calcium content and energy demand, whereas hibernation might be a kind of low demand-low supply situation with a low intracellular calcium level.