Inhibition of platelet aggregation by putrescine, spermidine, and spermine in hypercholesterolemic rabbits.

BACKGROUND: Hypercholesterolemia causes alterations in platelet function. Platelet hyperaggregation is considered a predisposing factor for atherosclerosis. In this paper, the antiaggregating effect of the polyamines putrescine, spermidine, and spermine was studied on platelets of normal and hypercholesterolemic rabbits. METHODS: New Zealand rabbits were fed with a cholesterol-enriched diet for 10 weeks. Lipids and glucose were determined in serum. The assays of platelet aggregation were carried out using platelet-rich plasma (PRP) obtained from both control and cholesterol-fed rabbits. We used 2.5 micromol /mL ADP and 2 microg/mL collagen as inductors of platelet aggregation. In addition, arginase activity and L-arginine content were determined in PRP. RESULTS: Serum total cholesterol and LDL-cholesterol concentrations were increased from 26.3 +/- 8.1 to 1,485.0 +/- 26.8 mg/dL and from 15.9 +/- 5.9 to 1,383.8 +/- 58.9 mg/dL, respectively, whereas triglyceride concentration increased from 88.3 +/- 35.6 to 411.0 +/- 154.5 mg/dL upon cholesterol feeding. Seventy-five percent of platelet aggregation inhibition was observed with 10 microM of polyamines in PRP of normal rabbits. Spermine inhibited platelet aggregation by 54% in PRP of hypercholesterolemic rabbits when ADP was used as agonist. The order of polyamine action was spermine > spermidine > putrescine. In addition, we found that platelet arginase activity and L-arginine content were unaltered upon hypercholesterolemia. CONCLUSIONS: These results show that the polyamines putrescine, spermidine, and spermine have antagonist action in platelet aggregation and suggest a key role of polyamines in platelet aggregation under normal and hypercholesterolemic conditions.

Vascular smooth muscle cells cultured on elastin membranes.

Smooth muscle cells from thoracic aortas of 12-week-old rats were cultured on elastin membranes for up to 21 days. The cell cultures were examined using light microscopy, transmission and scanning electron microscopy. The contractile phenotype characteristic for resident arterial wall muscle cell changed to the synthetic phenotype. In the synthetic state, the muscle cells contain few filaments, but a substantial amount of organelles are involved with synthesis. The cells grown on elastin substrates showed a multilayered pattern with the formation of nodules. Cell degeneration was present from day eight and increased with time. At the end of the experiment, the center of the multilayered areas showed degenerative changes with numerous foam cells of smooth muscle origin, areas of necrosis and a considerable amount of calcium deposit. Our experimental model would be valuable in the investigation of the pathological changes associated with smooth muscle cell proliferation in vessels.

Cell to stroma contacts in the tunica media of the hypertensive rat thoracic aorta.

The variation in mechanical stress to which the aortic wall is subjected requires that forces be transmitted between its components by means of relatively strong but compliant attachments. We have used transmission electron microscopy in order to study the cell to stroma contacts (smooth muscle cell-elastic fiber contact) in the tunica media of normotensive and hypertensive aortas of Sprague-Dawley rats. Hypertension was produced with a silver clip positioned around the left renal artery and the vessels were fixed by intravital perfusion at normal and elevated pressure. In ultrathin sections, the density of cell to stroma contacts per 100 microns cell perimeter and per 100 cell profiles were determined using an image analysis computer. In the hypertensive group the density of cell to stroma contacts fell considerably when compared with the control group. This research provides insights into the conditions under which high blood pressure may produce medial injuries and, perhaps, be a factor in the precipitation of dissections.

Myoendothelial contacts in the human fetal aorta.

Intracellular communication is an essential process in both embryological development and for the maintenance of normal tissue physiology. We have established the presence of this type of communication between endothelial cells and vascular smooth muscle cells in man and have here investigated the close contacts between endothelial and smooth muscle cells in the aorta of the 10-week-old human fetus. Transmission electron microscopy revealed myoendothelial contacts in the form of cytoplasmic projections, usually originating from the muscle cells. The nature of the cell-cell contacts was via simple appositions with an intercellular space of 6-15 nm. Myoendothelial contacts are sites for communication between elements of the intima and media, ensuring that paracrine secretion can occur and that the vessel wall, during growth and development, works as a unit.

The relationship between wall tension, lamellar thickness, and intercellular junctions in the fetal and adult aorta: its relevance to the pathology of dissecting aneurysm.

It is known that the distribution of stress and strain in the vessel wall is not uniform. We believe that this explains the location of the plane of dissection in dissecting aneurysms of large elastic arteries. We have investigated the effects of non-uniformity of stress and strain on the thickness of each elastic lamella and on the distribution of intercellular junctions in the media of developing and adult rats, to seek evidence to support this hypothesis. Intercellular junctions were identified by transmission electron microscopy of whole wall sections. A morphometric study of elastic tissue distribution was made on an image analysis computer. Differences were analysed using one-way analysis of variance. There are between six and eight elastic lamellae in the aorta of rats. In the fetus, only the internal elastic lamella is complete; the others were not fully formed by term. In the adult, the inner five elastic lamellae were thicker than the remaining two or three, and smooth muscle cells in the thicker lamellar units had more cell-cell contacts of all types examined. These data support the concept of a difference in stress-resisting properties of the aortic wall on the junctions between the inner two-thirds and the outer third of the media. The findings indicate that, as proposed in theoretical models the innermost lamellae support the high tension. In the adult aorta, the structure is modified to enhance the capacity to resist stress in the internal two-thirds of the media.(ABSTRACT TRUNCATED AT 250 WORDS)

Myoendothelial contacts in arteriolosclerosis.

Human renal biopsies were examined electron microscopically to investigate close contacts between endothelial and smooth muscle cells in small arterioles. These myoendothelial contacts were seen in the form of cytoplasmic projections passing through fenestrae in the basal lamina. Most of these cell processes seem to arise from the endothelial cells. In the control vessels, the separation between the endothelial cells and smooth muscle cells of the tunica media is 0.09-0.27 microns. With arteriolosclerosis there is an increasing separation between the elements of the intima and the media, from 1.0 to 2.42 microns. In spite of this increasing separation, myoendothelial contacts maintain an intercellular space of 10-15 nm, as observed in the control vessels. At 2.42 microns of separation, the amount of extracellular material accumulated is such that the cells can no longer keep in contact. Break up of the myoendothelial contacts may be responsible for impairment of communication between the tunica intima and media in the vessel wall in arteriolosclerosis.

Myoendothelial contacts in the thoracic aorta of rat fetuses.

There is increasing documentation of the presence of endothelial-smooth muscle communication in both small and large arteries. We have found that morphological evidence of these contacts appears early in development. Intimate contacts between endothelial and muscle cells in the thoracic aorta of normal rat fetuses have been studied with transmission electron microscopy. These myoendothelial contacts were seen in the form of cytoplasmic projections passing through fenestrae in the elastic lamellae. Most of these cell processes seem to arise from the muscle cells and they usually have a club-shaped configuration. The specific nature of the cell-cell contact was predominantly via simple appositions with an intercellular space of 6-15 nm. Myoendothelial contacts are potential sites for electrical and metabolic communication between elements of the intima and media, ensuring that the vessel wall works as a unit. The demonstrated necessity of intact endothelium for smooth muscle cell response to load may depend on this type of contact.

Intercellular contacts in the media of the thoracic aorta of rat fetuses treated with beta-aminopropionitrile.

Beta-aminopropionitrile (BAPN) alters the scleroproteins of the arterial media in a way which permits an increase in the stress (force/unit area) applied to smooth muscle cells at a given pressure. Pregnant Sprague-Dawley rats were treated with BAPN and the thoracic aortas of exposed fetuses were studied by electron microscopy and image analysis. Transmission electron microscopy of the media of the thoracic aorta revealed that the types of intercellular contacts seen in the controls and in the BAPN group were intermediate junctions (IJs), nexus junctions (NJs), simple appositions (SAs), and interdigitations (Ids) as in adult vessels. The BAPN-treated animals showed an increased density of IJs, SAs, and Ids when compared with controls but the NJs demonstrated no change. Load-sensitive intercellular contacts have been considered to be the major route by which stress is applied across cells, and the increased number in the BAPN group probably represents a response of the vessel wall to increased tangential tension.

Effects of hypertension on the intercellular contacts between smooth muscle cells in the rat thoracic aorta.

A qualitative and quantitative study was made of the contacts between muscle cells in the media of the thoracic aorta of hypertensive and normotensive Sprague-Dawley rats. In ultrathin sections, the type and number of contacts per 100 microns cell perimeter and per 100 cell profiles were determined using an image analysis computer. The intercellular contacts in both groups were in the form of simple appositions, interdigitations, intermediate junctions and nexus junctions. In the hypertensive group, there was a reduction in the number of simple appositions and intermediate junctions, but the nexus junctions (which provide intercellular communication) were increased in density. Interdigitations demonstrated no change. Decreased intercellular cohesion may play an important role in the mechanisms by which hypertension predisposes degeneration of the media with progression to ectasia, aneurysm formation and dissection.

Purkinje cell toxicity of beta-aminopropionitrile in the rat.

Compounds causing neurolathyrism are putative aetiological agents in neurodegenerative disorders including amyotrophic lateral sclerosis. beta-Aminopropionitrile (BAPN) is one such compound. We have administered this lathyrogenic agent at a dose of 1 g/kg by the intraperitoneal route in experiments in adult Sprague-Dawley rats during a period of 10 weeks. The rats developed marked kyphoscoliosis, ataxia with paralysis and muscle wasting of the hind limbs. Vacuolation and loss of Purkinje cells developed, but no anterior horn cell degeneration was noted. Immunohistochemical studies of phosphorylated neurofilaments and the 72 kDa heat shock protein were normal and no intraneuronal ubiquitinated inclusions were seen. High-dose intraperitoneal BAPN in the rat causes Purkinje cell changes, but no other central nervous system abnormalities.

Nexus junctions between vascular smooth muscle cells in the media of the thoracic aorta in normal and hypertensive rats. A freeze-fracture study.

Freeze-fracture electron microscopy and image analysis have been used to study the nexus junctions (NJ) between smooth muscle cells in the media of the thoracic aorta of normal and hypertensive Sprague-Dawley rats. The NJ found in both groups revealed particles on the P-face and depressions or pits in the E-face. Within each face, particle- or pit-free areas were identified in some junctions. NJ are usually surrounded by a narrow area devoid of particles and in certain fracture planes a close relationship of NJ with smooth endoplasmic reticulum was seen. Larger and more irregular NJ were found in the hypertensive group than in the controls. The differences observed in the hypertensive animals may be associated with an increase in tangential tension in the vessel wall.

Contact relationships between vascular smooth muscle cells: an in vivo and in vitro study.

Cell/cell contacts in the intact media of the rat thoracic aorta and inferior vena cava, and in cultured smooth muscle cells from these vessels have been quantified in order to determine whether their density and their type can be correlated with the differences in load at these two sites. The type and number of contacts per 100 microns of cell perimeter and per 100 cells were determined with a semi-automatic image analysis system. In all cases there was a predominance of simple appositions; intermediate junctions, interdigitations, and nexus junctions were less frequently observed. There were more cell contacts of each type in the intact arteries than in the intact veins. In cell cultures, all types of cell contacts were increased but the proportion of load bearing types was reduced.

Myoendothelial contacts in the small arterioles of human kidney.

Human renal biopsies were examined electron microscopically to investigate the intercellular relationships between endothelium and smooth muscle in renal arterioles. Three types of contacts were seen occurring through perforations in the basal lamina. These were classified by their origin either, from an endothelial cell, from a smooth muscle cell or from both. The cell processes involved were usually irregular but some were identified as club-shaped. The nature of the contacts was predominantly via simple appositions with an intercellular space of 6.9-13 nm. These myoendothelial contacts may play an important role as sensors of hypertensive load.

Intercellular contacts in tumours of the vascular smooth muscle cells in man.

Electron microscopy has been used to study the cell to cell and cell to stroma contacts between smooth muscle cells (SMC) in normal vessels, angiomyolipoma and well and poorly differentiated vascular leiomyosarcoma. Micrographs were examined with a semiautomatic image analysis system. The length of the cell borders was calculated and the type and number of contacts per 100 micron cell perimeter and per 100 cells were determined. In all cases there was a predominance of simple appositions. Intermediate junctions, nexus junctions, interdigitations, intermediate contacts and junctional interdigitations were less frequently observed. In general, as the SMC become malignant and less differentiated the number of cell to stroma attachments decreases markedly and the intercellular contacts increase slightly.

In-vitro demonstration of a complicated atherosclerosis-like lesion.

Smooth muscle cells derived from the media of adult rat aorta were grown on elastin membranes for up to 3 weeks in cell culture. Cell degeneration was evident by the 4th day and increased with time. At the end of the experiment necrotic and calcified areas were prominent. The basic feature of cell degeneration was dissolution of myofilaments; the disintegrating cytoplasm usually contained numerous glycogen granules. Increased electron density of the mitochondrial matrix and fat globules were seen and nuclei showed dilution of chromatin or pyknosis. Abundant cellular debris, vesicular structures and microfibrils were found in the extracellular space.