Regression of cholesterol induced venous plaques after cholesterol withdrawal in rabbits.

Rabbits receiving a dietary cholesterol supplement of 0.5% develop atheromatous plaques in the systemic arteries, the pulmonary artery, the pulmonary veins and the venous cavities of the plexus pampiniformis in the funiculus spermaticus. Six months after the withdrawal of the cholesterol supplement the arterial lesions are still present, and show a fibrous transformation. This study is the first report of the total regression of the lesions in the particular venous localizations of the lungs and the plexus pampiniformis. The level of intraluminal pressure is discussed as the possible mechanism responsible for the diverging vascular reactivity.

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.

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.

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.

Centrolobular liver fibrosis in the hypercholesterolemic rabbit.

During a study on the development of atheromatous lesions in rabbits fed a diet with a low or high cholesterol supplement, we found a moderate to pronounced centrolobular liver fibrosis. This fibrosis developed in three stages. Early after supplementation of cholesterol, we observed increased immunoreactivity of collagen types I, III, and IV, and fibronectin, around central veins and in adjacent sinusoids. In the second stage, we observed further increase of collagen and fibronectin immunoreactivity, together with the appearance of alpha-smooth muscle actin (alpha-SM actin)-positive cells and anti-rabbit macrophage monoclonal antibody (RAM 11)-positive cells. In the third stage, we observed large numbers of alpha-SM actin-positive cells, together with heavy deposition of connective tissue proteins in pericentral sinusoids, in addition to focal atrophy of parenchymal cells. By transmission electron microscopy (TEM), fat-storing cells in the pericentral regions were shown to be enlarged, to lose their lipid-droplets, and to acquire dilated rough endoplasmic reticulum corresponding to an activated phenotype. Parenchymal cells were either normal or contained numerous small lipid-droplets. They sometimes were smaller and distorted. Northern hybridization performed on total RNA of whole liver showed an increased level of collagen alpha1(I), alpha1(III), and alpha1(IV) messenger RNA (mRNA) after 24 weeks of low dietary cholesterol supplementation. These data show enhanced expression of extracellular matrix proteins. We conclude that cholesterol overload induces pericentral liver fibrosis in rabbits. The diet clearly activates fat-storing cells to become fibrogenic effector cells. At present, we have no explanation why hypercholesterolemia induces phenotypic transition of fat-storing cells.

Destruction of lacis cells of the juxtaglomerular apparatus in the hypercholesterolemic rabbit.

During a study of experimentally induced atheromatosis in hypercholesterolemic rabbits we found a heavy infiltration of the lacis cell field (G field) by lipids in the kidney. A systematic study was done in 12 rabbits receiving a dietary cholesterol supplement of 0.3% for 26 weeks and in 8 animals receiving a supplement of 2% for 8 weeks. Standard histology, immunohistochemistry for a-smooth muscle cell actin and with the macrophage specific antibody RAM 11, and transmission electron microscopy were performed. In the low cholesterol, long duration group, 90% of the G fields were involved. The lipid accumulation was mainly extracellular. The lacis cells contained moderate numbers of medium sized lipid vacuoles and showed signs of cytoplasmic damage leading to disintegration. The mesangial cells contained few dipid droplets and the matrix was normal. The arterioles were not involved. The lesion suggests an impairment of the clearance of lipids, leading to interstitial accumulation and cellular damage.

The influence of collapse of the lung parenchyma on the morphology of pulmonary blood vessels in the rat.

During a search for resident subendothelial smooth muscle cells in pulmonary vessels of the rat we found that in expanded lungs the muscular pads in the veins, considered by some authors as sphincters, were hardly visible whereas in collapsed lungs they were very conspicuous. In a separate study intended to quantify the degree of collapse or expansion the left lung was examined in 5 rats with a collapsed and in 5 rats with an expanded lung: the expansion was produced by filling the airways by gravity with Methacarn fixative. The degree of expansion was determined by morphometry measuring the volume density of the tissue fraction of the pulmonary parenchyma in the microscopic sections: in the expanded lung the mean value was 8.5% (range 6.7-12.6%), in the collapsed lung 20.1% (range 18.7-22.3%), a highly significant difference (p < 0.000). Serial sections generally 60-100, 6-microns-thick, were stained by PAS, Sirius red hematoxylin and Verhoeff's elastic stains. Immunohistochemical staining was done with monoclonal antibody against alpha smooth muscle cell actin and desmin. Graphic reconstructions of representative vessels were performed. It was shown that the muscular media of the veins was interrupted and that the muscular pads corresponded to the contracted smooth muscle cell segments alternating with the noncontracted segments devoid of muscle. In the expanded lungs muscular pads were flattened and often hardly detectable. This indicates that the structures considered as sphincters are postmortem contraction rings in collapsed lungs.

Sephadex induced granulomatous reaction in rats.

Granuloma formation in the rat lung after single or repeated i.v. injections, intratracheal instillation and intradermal implantation of Sephadex beads was studied over a time period from 3 h to 3 mo. Macrophages were identified with the mAB ED1, vascular smooth muscle cells with an mAB against alpha SMC-actin, endothelial cells with a polyclonal AB against factor VIII and cyclic activity with an mAB against PCNA. The morphology and the time course of the development of the granulomas is identical in the different experimental conditions. The macrophages form the bulk of the cellular infiltrates, giant cells appear after 24 h. Cyclic activity is early and marked in the interstitially located macrophages, it is delayed and slight around the beads, suggesting a biphasic pattern. The macrophage reaction is markedly enhanced after a second i.v. injection, indicating the development of a hypersensitivity state. Numerous eosinophils are located in the interstitium, but only few are in direct contact with the beads. Their number doesn't increase after a second i.v. injection. Intradermal implantations show only macrophages and lymphocytes. A special feature is the disappearance of the arterial wall around the beads resulting in extrusion without haemorrhages or thromboses.

Hyperplasia, hypertrophy, and phenotypic alterations in the distal nephron after acute proximal tubular injury in the rat.

BACKGROUND: Little is known about the impact of acute proximal tubular injury and dysfunction on the distal nephron. EXPERIMENTAL DESIGN: Selective necrosis of the kidney proximal convoluted tubule (PCT) was induced in rats by subcutaneous injection of the aminoglycoside gentamicin during 2 days. Damage and repair were measured until complete morphologic recovery after 10 days. Special attention was given to structural and biochemical alterations in the distal nephron. RESULTS: In control animals, cellular turnover, measured by immunohistochemical staining for proliferating cell nuclear antigen, was higher in distal than in proximal tubules. After injury, the strongly increased cell proliferation in regenerating necrotic PCT was preceded by an equally important proliferation in the distal tubules of the cortex and outer stripe of the outer medulla in the absence of necrosis but displaying enhanced apoptosis. Yet, epithelial vimentin expression was restricted to regenerating PCT. A temporary loss in the amount of immunostainable epidermal growth factor in the distal nephron was paralleled by a similar reduction in Tamm- Horsfall protein and transferrin receptor staining and in peanut and Helix pomatia lectin binding. Furthermore, the epithelial area/nucleus in the cortical distal tubules was increased by 71%, 6 days after the onset of acute renal failure; this hypertrophic condition was confirmed ultrastructurally. After full recovery of the PCT, a second burst in proliferative activity occurred in the hypertrophic distal segments in the absence of apoptosis. In the regenerated PCT, an excess cell number was accompanied by increased apoptotic activity. CONCLUSIONS: Development of distal tubular hypertrophy after PCT necrosis may be a compensatory response to a transient loss of proximal tubular function. The early reduction in staining for epidermal growth factor and other distal tubular markers in the presence of apoptosis and hyperplasia indicates transient phenotypic simplification and implies that renal epidermal growth factor is unlikely to control PCT regeneration.

The relationship between pre-existing subendothelial smooth muscle cell accumulations and foam cell lesions in cholesterol-fed rabbits.

We investigated whether pre-existing subendothelial smooth muscle cell (SMC) accumulations in cholesterol-fed rabbits are transformed into foam cell plaques. Twenty-four rabbits received a standard diet supplemented with 2% cholesterol for 4 or 8 weeks. Six rabbits received a supplement of 0.3% cholesterol for 35 weeks. The aorta and other systemic and pulmonary vessels were studied by immunohistochemistry for smooth muscle cells SMC (alpha-SMC actin), macrophages (RAM11), cell replication (proliferating cell nuclear antigen) and endothelial cells (von Willebrand factor; vWF). Initially the foam cell plaques were composed exclusively of foam cells of macrophage origin (MFC). In more advanced lesions SMC and collagen fibres were also present, leading to a fibrous transformation of the plaque. Cell replication was mainly located in the MFC. The endothelial cells covering the plaques showed an increased immunoreactivity for vWF which was also deposited in the interstitium between the FC. Pre-existing subendothelial SMC did not transform into FC. The newly formed FC plaques remained clearly separated from the pre-existing subendothelial SMC. The development of the plaques can be attributed not only to monocyte recruitment but also to macrophage multiplication.

Ultrastructural demonstration of type IV collagen deposits in periductal elastosis in breast cancer.

In a previous study we demonstrated at light microscopical level the presence of variable amounts of type IV collagen in the areas of periductal and interstitial elastosis in breast cancer. The present work was directed towards a further study by immunoelectron microscopy of the distribution of type IV collagen in the areas of periductal elastosis. The semithin sections showed a distinct immunoreactivity of all basement membranes for type IV collagen but no staining of the interstitial stroma. Corresponding ultrathin sections demonstrated a broad basement membrane with immunoreactivity for type IV collagen at its outer side. Many punctiform deposits of type IV collagen were observed in the areas of periductal elastosis but not around normal ducts or vessels. Recently the role of type IV collagen as a structural component on anchoring plaques between the basement membrane and the underlying stroma in the dermis has been emphasized. The results of this study demonstrate the presence of type IV collagen deposits below a thickened basement membrane in breast cancer.

Longitudinally oriented smooth muscle cells in rabbit arteries.

Intima formation in vessels, spontaneous or experimentally induced, is generally characterized by the presence of longitudinally oriented smooth muscle cells (LSMC). During an experiment of neo-intima induction in carotid arteries in rabbits, by application of a non-constrictive silastic cuff, a study was performed to investigate the presence of LSMC in the systemic and pulmonary circulations, in both elastic and muscular arteries. Three patterns could be distinguished: intimal cushions in muscular arteries, single or small groups of LSMC in the intima in elastic and larger muscular arteries, and intra-medially located layers or columns of LSMC in the aorta, the pulmonary artery, at the bifurcation of the aorta and around orifices of branches. In order to understand this peculiar orientation a biomechanical approach was used: this showed that near the lumen the circumferential stress is 4.5 times higher than the longitudinal. Because the cell surface of the smooth muscle cells exposed to this stress per unit vessel length is much less in the longitudinal than in the circular direction we conclude that the LSMC align in the direction which allows them to cope most effectively with the mechanical stresses.

Ileal intussusception due to invagination of Meckel's diverticulum.

A middle-aged patient is reported who came to laparotomy after a six months period of abdominal complaints. Extensive investigations couldn't explain his symptoms. At laparotomy he was found to have an ileal intussusception due to an invaginated Meckel's diverticulum. These events are confronted with other collected literature reports. Symptomatology and treatment are discussed.

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.

Effect of ageing and malnutrition on rat myocardium. II. The microvasculature.

The modulating effects of ageing and malnutrition on rat myocardium were studied morphometrically with respect to the microvasculature. An increase in capillary density together with a decrease in capillary lumen cross-sectional area was noted during starvation. The important changes seen in the myocyte T-system were paralleled by a decreased diffusion distance for oxygen from the capillary lumen to the mitochondrion. The changes described in the aged rat heart point to an altered inter-relationship between parenchyma and vascularization with a lower capillary volume fraction and a greater diffusion distance from the capillary lumen to the mitochondrion; this is caused by hypertrophy of the aged myocyte. This reduction in capacity to exchange substrates is further reduced by the less developed T-system in the older myocyte.

Quantitative relationships between body weight, kidney weight and nephron size in mongrel dogs.

Mammalian species of large stature have larger kidneys with larger nephrons than smaller animals, although this relationship is not linear. In order to investigate whether in animals of different sizes within the same species similar differences exist, a study in mongrel dogs was undertaken. In animals weighing between 9 and 42 kg with kidney weights from 25 to 79 g the area and perimeter of the glomeruli and the cortical width were measured. The number of glomeruli per unit area was counted and corrected for the size of the glomeruli. The results show that larger dogs have larger kidneys and that this enlargement is due to the increase in size of the nephrons and not in their number.

A standardized surgical technique to obtain a stable and reproducible chronic renal failure model in dogs.

The remnant kidney, a model of chronic renal failure in animals, can be obtained by two techniques: either surgical removal of tissue of one kidney, combined with contralateral nephrectomy or inducing necrosis of kidney tissue by ligation of branches of the renal artery of one kidney combined with contralateral nephrectomy. In the literature, most reports concern the ligation technique. The technique is safe and simple but the results in dogs are unpredictable. In this paper, both techniques were compared. We could demonstrate that the unpredictable result of the ligation technique is due to the formation of collateral vessels bypassing the ligated branches and to the inconstant ramification pattern of the renal artery. In this study, a standardized technique consisting of the resection of 16-18 g of tissue of one beagle kidney and removal of the other one is described. This method results in a stable chronic renal failure until the dogs are sacrificed at 9-12 months.

Stromal populations and fibrosis in human long-term bone marrow cultures.

An immunofluorescence study of the adherent layer of human long-term bone marrow cultures (HLTBMC) revealed the following surface markers on the different stromal cell populations: stromal fibroblastic cells CD10+, FIB86.3+, CD13+, CD71+; adipocytes CD10+, FIB86.3-, CD13+, CD71-/+; and macrophages CD10-/+, FIB86.3+, CD13+, CD71-/+, CD14+, CD33+, CD25+, HLA-DR+, CD4+, CD19+, CD45+. The markers of the stromal fibroblastic cells in HLTBMC were similar to those of twice-passaged fibroblasts not only from bone marrow and spleen, but also from a hemopoietic non-supportive organ such as the skin. Some of the cultured human umbilical vein endothelial cells used as controls were found to be CD25+, demonstrating for the first time the interleukin-2 receptor p55 chain on normal non-hemopoietic cells. The stromal fibroblastic cells are overrepresented compared to the small non-macrophage hemopoietic cell population in the adherent layer of HLTBMC. In addition, silver staining revealed an increased reticulin content in most of the HLTBMC. An excessive growth of stromal fibroblastic cells and an excessive deposition of their product, the reticulin fibers, are the hallmark of myelofibrosis. The finding of equivalent observations in HLTBMC suggests that the hitherto unexplained, premature quenching of hemopoiesis in HLTBMC might at least partly be due to mechanisms similar to those operating in myelofibrosis in vivo.

Ridogrel prevents the thromboxane-mediated pressor response and oedema induced by hydrogen peroxide in isolated rabbit lungs.

Perfusion of isolated rabbit lungs with hydrogen peroxide (H2O2, 3 x 10(-5) M) raised the overflow of thromboxane B2 (TXB2) and the perfusion pressure. H2O2 induced oedema formation and endothelial distress, as evidenced by an increased production of 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha). Endothelial cell death did not occur since there was no release of lactate dehydrogenase. The thromboxane A2 (TXA2)-synthase inhibitor/receptor antagonist ridogrel (R68070) further enhanced 6-oxo-PGF1 alpha output, while inhibiting TXB2 release. Ridogrel prevented the rise in pulmonary artery pressure and oedema formation. These data indicate that TXA2 is probably involved in the acute pulmonary pressor response and concomitant oedema formation induced by H2O2. In order to assess the functional activity of the pulmonary endothelium, the uptake of 5-hydroxytryptamine (5-HT) was measured before and 15 min after exposure to H2O2. As the H2O2-induced effects were not associated with any change in the uptake of 5-hydroxytryptamine (5-HT), we conclude that the endothelial injury was reversible or that the 5-HT uptake was not sensitive enough to evaluate the integrity of the pulmonary endothelium during oxidant-induced injury.

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.