Norbert Buyssens

Cell Composition, Replication, and Apoptosis in Atherosclerotic Plaques After 6 Months of Cholesterol Withdrawal

Unstable human atherosclerotic plaques are characterized by a thin fibrous cap that contains few smooth muscle cells (SMCs) and numerous foam cells of macrophagic origin. Apoptosis of SMCs in the fibrous cap could destabilize the plaque and promote plaque rupture. In an experimental approach, we have studied apoptotic cell death and related proteins in atherosclerotic plaques of cholesterol-fed rabbits and examined the effects of cholesterol withdrawal. The induced atherosclerotic plaques at the thoracic aorta were composed of both fibromuscular tissue and foam cells. The presence of SMCs overlying macrophage accumulation was reminiscent of the structure of human atherosclerotic plaques. The plaques showed signs of cell replication and apoptotic cell death (1.8+/-0.5% terminal deoxynucleotidyl transferase end-labeling [TUNEL]-positive nuclei). Cell replication was confined mostly to the macrophages, whereas 34% of the TUNEL-labeled cells were SMCs. Both the macrophages and SMCs in the plaques expressed BAX, a proapoptotic protein of the BCL-2 family. After 6 months of cholesterol withdrawal, the thickness of the plaques in all localizations of the aorta was unchanged, but apoptosis was nearly absent (<0.1% of nuclei). Moreover, macrophages disappeared from the plaques, whereas the SMCs that remained present lost their lipid accumulation and strongly reduced their BAX expression. These changes were associated with a reduction of cell replication and increased deposition of fibrillar collagen fibers in the plaques, which pointed to plaque stabilization. In conclusion, the cell composition but not the thickness of atherosclerotic plaques was profoundly altered after a 6-month cholesterol withdrawal period. These changes were associated with a strong reduction of cell replication and apoptotic cell death. Moreover, the expression of the proapoptotic factor, BAX, was reduced in the remaining cells, which were mainly SMCs. These findings could help to explain the benefit of lipid-lowering therapy on plaque stabilization.

Possible Mechanisms of Collar-Induced Intimal Thickening

The positioning of a soft silicone collar around the rabbit carotid artery induces intimal thickening. We investigated to which extent occlusion of the vasa vasorum, damage of the perivascular nerve network, and/or changes in blood flow velocity contribute to intimal thickening. To this end, collars with different bores (diameter of inlet and outlet) were positioned around the carotid artery of male rabbits for 14 days. In another experiment, 75% of the wall of fitting collars was removed (open collar). In the midcollar region, the cross-sectional area of the intima reached a maximum (72 +/- 14 mm2/1000) when the endings of the collar fitted the artery closely. Removal of the side wall of these fitting collars reduced intimal thickening by 90%. Examination of unoperated carotid arteries never showed penetration of the adventitia or the media by vasa vasorum. The perivascular neuronal network in the region surrounded by a closed or an open collar was almost completely lost as compared with the zones outside the collar. Both the closed and open collar slightly bent the artery and increased the peak systolic velocity, measured with pulsed color Doppler after 6 hours, to a similar extent as compared with the proximal zone outside the collar. After 2 weeks, the peak systolic velocity within both the closed and open collar was partly normalized and was statistically not different from the proximal zone outside the collar. In conclusion, the geometry of the collar influenced the extent of intimal thickening, whereby more intimal thickening was obtained with a collar whose endings fit the carotid artery, rather than with a loose collar. Moreover, a closed structure was essential. The results obtained with the open collar exclude occlusion of vasa vasorum, damage of the perivascular neuronal network, kinking of the artery, and changes in blood flow velocity as major factors in the collar-induced intimal thickening. Our findings are consistent with the possibility that intimal thickening is the consequence of the combination of both vascular injury and hindrance of transmural flow by the collar. The obstruction of transmural fluid transport may then lead to retention of toxic metabolites, and/or cytokines within the segment enclosed by the collar.

Chronic myelocytic leukemia versus idiopathic myelofibrosis. A diagnostic problem in bone marrow biopsies

Repeated bone marrow biopsies of 12 patients with chronic myelocytic leukemia (CML) and 13 patients with idiopathic myelofibrosis (IMF) were compared. The amount of cellularity, the composition of the cell population, and the stromal changes were assessed. Both diseases were divided into four stages, each with a consistent morphologic pattern. In CML, Stage 1 presented a tremendous increase in granulocytes and a drop of the erythroid series; Stage 2 showed a shift to myelocytes and a moderate increase of blasts; in Stage 3 there was a strong increase of blasts and the reappearance of red cells; and in Stage 4 hematopoietic cells disappeared with metastases of abnormal blasts to many organs. The stroma showed a progressive fibrosis so that by Stage 4 there was a totally fibrotic marrow. With IMF, Stage 1 presented a dysharmonic trilinear proliferation; in Stage 2 there was a maximum of cellularity, which remained trilinear; in Stage 3 there was a decrease of hematopoietic cells; and in Stage 4 these cells disappeared, but hematopoietic cells in dilated sinusoids of the empty marrow became apparent. The stroma showed a progressive fibrosis, ending by Stage 4 in a totally fibrotic marrow indistinguishable from the similar stage of CML. Careful analysis of the bone marrow biopsy findings permitted the stage of the disease and hence the prognosis to be determined; Stage 3 for CML means death within a short period of time. In clinically “acute” myelocytic leukemia, the biopsy helps to make the distinction between Stage 3 of an unsuspected CML and acute myelocytic leukemia.

Neointima formation impairs endothelial muscarinic receptors while enhancing prostacyclin-mediated responses in the rabbit carotid artery.

The purpose of this study was to determine whether the generation of a neointima, an early step in the development of atherosclerosis, affects endothelium-dependent or -independent vasodilation. The neointima was induced, within 7 days, by positioning a nonocclusive silicone collar around one carotid artery in rabbits. After 1, 2, 7, or 14 days segments were cut from the collar-surrounded region of this artery as well as from the sham-operated contralateral artery and were used for isometric tension recording or for bioassay of nitric oxide (NO). The acetylcholine-induced release of NO was significantly reduced at 7 days. The tension recordings suggested that this already occurred at the earliest stages of neointima formation. Neither the capacity of the endothelial cells to form NO in response to the calcium ionophore A23187 nor the capacity of the underlying smooth muscle cells to relax in response to sources of exogenous NO (3-morpholinosydnonimine and nitroglycerin) was affected by the neointima. Therefore, the impaired endothelium-dependent relaxations to acetylcholine are presumably due to a defect at the level of the endothelial muscarinic receptors. The presence of a fully developed neointima did not alter the responsiveness to isoproterenol and forskolin but enhanced prostacyclin-mediated responses (assessed by iloprost and 13-hydroxyoctadecadienoic acid). These results illustrate selective alterations of endothelial and smooth muscle cell function in intima generation before fatty streak formation.

Localization of extracellular superoxide dismutase in rat lung: neutrophils and macrophages as carriers of the enzyme

Immunohistochemistry (IHC) and in situ hybridization (ISH) was used to localize extracellular superoxide dismutase (EC-SOD) and its mRNA in rat lung before and after a lipopolysaccharide (LPS)- and hyperoxia-induced inflammation. In control rats, EC-SOD mRNA was synthesized in macrophages and in cells of the arterial vessel walls and the alveolar septa. The EC-SOD protein was mainly localized in plasma and on the apical side of the epithelial cells located near bronchus-associated lymphoid tissue (BALT). ISH did not reveal major changes in the distribution of EC-SOD mRNA upon induction of inflammation. In contrast, IHC demonstrated a progressive staining of the epithelium of the larger bronchi for the protein. Neutrophils and macrophages invading the lung showed an intensive staining for the EC-SOD protein concomitantly with a decrease of the enzyme in the plasma. Twenty-four hours after LPS stimulation only a spotty positivity remained on neutrophils in and between the alveolar spaces. In the bronchoalveolar lavage fluid (BALF), only macrophages showed a strong positivity for EC-SOD mRNA while the protein was detected in macrophages and neutrophils. Exposure to hyperoxia did not affect the distribution of EC-SOD mRNA and protein. The presented data demonstrated that in lung tissue the EC-SOD enzyme may have a protective function for activated macrophages, neutrophils, and lympoid tissue-associated epithelial cells.

Nevus cell maturation or atrophy

Nevus cells show considerable variation in their appearance, depending on their localization in epidermis, upper dermis, or deep dermis. The difference in appearance of nevus cells when located superficially or in the deep dermis has been referred to as nevus cell “maturation.” The aim of this study is to objectify morphological differences between nevus cells in epidermis, upper dermis, and deep dermis by means of a morphometric study at light- and electron microscopic levels. The results show that there is a decrease in number and size of all structures except for mitochondria and microfilaments. These findings are consistent with atrophy. The concepts of maturation, differentiation, and atrophy are also discussed.

Comparison of cell growth in different parts of breast cancers

This study was performed to answer the question: which parts of breast cancers are active in terms of proliferation as measured by the Ki‐67 antibody and in terms of cell division as measured by the mitotic index. Forty‐six breast samples were studied, including 34 breast cancers and 12 benign conditions. The intraductal component of infiltrating breast cancers showed a significantly lower proliferation index than the infiltrating component. The cells at the periphery of infiltrating tumour strands showed a higher proliferation activity than the cells in the core. These findings suggest that infiltration advances through preferential active growth of the cells at the invasion front.

Heat-stable alkaline phosphatase as a marker for human and monkey type-I pneumocytes

SummaryThe expression of the heat-stable isoenzyme of alkaline phosphatase in the human and monkey (Macaca mulatta, M. fascicularis) lung was investigated at the light- and electron-microscopic level, using cytochemical techniques and immunocytochemical procedures based on monoclonal and polyclonal antibodies against human term-placental alkaline phosphatase. Both in man and monkey, the enzyme was present in type-I pneumocytes. In the monkey, the enzyme was found in all type-I cells. In man, strong staining was observed only in some type-I cells and in certain cuboidal respiratory bronchiolar cells. Staining was localized on the apical and basal plasma membrane, in apical and basal caveolae, and in the underlying basement membrane. The level of heat-stable alkaline phosphatase expression in the human lung was 10-fold lower than in the monkeys studied. In human fetal lung, the onset of heat-stable alkaline phosphatase expression was associated with the development of the alveolar epithelium from 17–20 weeks gestation onward. It is concluded that: (1) heat-stable alkaline phosphatase is a specific constitutent of type-I pneumocytes in man and monkeys; and (2) its subcellular localization may explain its rapid appearance in the circulation under certain conditions.

Desmin-positive stellate cells associated with angiogenesis in a tumour and non-tumour system.

SummaryThe angiogenesis induced after implantation of fragments of the Walker 256 carcinoma was compared with the angiogenesis following implantation of different amounts of Indian ink. Morphologically and chronologically the tumour system showed no difference from the Indian ink system, provided sufficient amounts of ink were implanted. Both systems were characterized by significant macrophage infiltration. The vascular development, which was clearly concentrated in a dense rim around the tumour, remained present when the tumour enlarged, suggesting an acquisition of vasculature by the tumour through vessel incorporation and not vessel ingrowth. Initially, scattered desmin-positive cells, in contact or encircled by collagen IV, were found in the developing angiogenic rim. Later many desmin-positive cells were found around vessels and could be identified by electron microscopy as pericytes. They exhibited close local contacts with endothelial cells. After incorporation of the peritumour vascular rim into the tumour the number of pericytes decreased and their shape became flattened and elongated.

Effect of ageing and malnutrition on rat myocardium. I: The myocyte

The effects of ageing and starvation on the rat myocardium were studied by morphometric methods. Since cardiac muscle is a tissue with a high level of anisotropy, methods based on the concept of vertical planes were used to describe quantitative alterations in the rat myocyte both at the cellular and ultrastructural level. During starvation rapid and important changes were noted, particularly in the transverse dimension of cells and organelles. The most striking change, however, was the immediate dilatation of the myocyte T-system, reflecting an adaptive interaction between the intra- and extracellular environment. At the same time exocytosis of intracellular components into the extracellular space of the T-system was observed. The ratio of mitochondria to myofibrils decreased progressively during starvation. Such a decrease, in general, may reach a point when cellular energy supply becomes compromised. A comparison between different regions of the heart showed no differences and it can be concluded that the morphological changes during starvation are the same, and equally distributed, in both ventricles. The changes described in the aged rat heart point in the direction of a hypertrophy of the aged myocyte. This leads to a lower ratio between surface and volume which finds its representation at the subcellular level in a more spherical shape of nuclei and mitochondria. Unlike what is seen in malnutrition, the mitochondrial/myofibril ratio is higher in the older rat. From the morphological point of view, the atrophy of malnutrition and the hypertrophy of ageing are opposed, but in both there is a change in the relationship of the myocyte to its environment which directly influences the substrate exchange capacity. This tends to protect the myocyte in starvation but jeopardizes the older cell.