Vascular smooth muscle cells are of major interest for the evaluation of atherosclerotic processes. By the use of two-dimensional gel electrophoresis and subsequent silver staining we investigated the protein expression of smooth muscle cells in the native thoracic aorta, immediately after enzymatic disaggregation, in subconfluent proliferating and in postconfluent nonproliferating primary cell cultures. Compared to the native thoracic aorta the protein composition of smooth muscle cells in cell culture is changed dramatically. Furthermore, significant differences in protein expression between proliferating and nonproliferating smooth muscle cells in cell culture were found.

Animals↗

PubMedDate not supplied

Quantitative measurement of local blood flow with heat clearance.

Measurements of the local blood flow through organs by means of heated thermistor-probes enable one to perform continuous recordings of flow. The principle is based on the measurement between a heated and an unheated probe, both implanted in the tissue or placed on the superficial layer of the organ. The flow-measure for this type of blood flow recording is the heat transported from the heated site of the tissue. It is measured in cal-cm(-1)-sec(-1)-degree C(-1). This is the dimension of the heat conductance. Quantification of continuous blood flow measurements in ml flow/g tissue-min has been carried out with combinations of gas clearance and heat transport. It has now become possible to measure blood flow in ml blood-ml(-1) tissue-min(-1) solely with the aid implanted thermistors. Two types of local blood flow measurements are described. 1. Measurements with "slug heating" by which a thermistor is heated for a very short time and the temperature change is subsequently recorded. 2. The measurement of flow by sudden introduction of heat, if a heating coil around a micro-thermistor is switched on or off and the course of the tissue temperature is subsequently followed. It is proven, that the temperature of a point-shaped micro-thermistor in nonperfused tissue and in homogeneous perfused tissue differs only by an exponential factor (see article) where uslug is the temperature field after a slug injection of heat dependent on location, time, blood flow (phi) and partition coefficient for heat (lambda) and where index 0 = unperfused tissue. If the heater is not heated in the form of a delta function but is switched on and the temperature is subsequently recorded or if the heater, after a period of constant temperature, is switched off, the derivatives in respect to time differ only by an exponential factor and can be used as a measure of flow. The evaluation of blood flow is described in detail. The measurements show the following advantages, compared with former techniques: 1. Heat clearance and continuous blood flow measurements by means of heat transport measurements are both possible at the same site. 2. No other operation and procedures are necessary except implantation of the thermistor. 3. Turning on the heater does not affect the tissue and does not cause emotional reactions. As examples the measurements of local blood flow in brain tissue and in the myocardium of conscious animals are described.

Animals↗

PubMedDate not supplied

Effect of the calcium entry blocker, flunarizine, on ruthenium red uptake by endothelial cells following acute electrical stimulation of rabbit carotid arteries.

Local transmural electrical stimulation with DC of a carotid artery by means of implanted electrodes causes subendothelial fibromuscular proliferates or atheroma (if the animal receives a cholesterol-containing diet) beneath the anode. The endothelial lining is maintained when weak current is used for stimulation. The model permits studies of permeability of the endothelium in all stages of the plaque development. Ruthenium red as a marker for the glycocalyx is transiently taken up into the cytoplasm of the endothelial cells beneath the anode immediately after a 30 or 60 min lasting stimulation period. When staining the endothelium later than two hours after the end of an acute stimulation period, the ruthenium red staining is again normal. This indicates that the increased permeability to large molecules is reversible. Injection of the calcium entry blocker Flunarizine inhibited the cytoplasmic uptake of ruthenium red, showing that an increased entry of calcium into the endothelial cells may contribute to the disturbance in the permeability of large molecules into the endothelium.

Animals↗

PubMedDate not supplied

Responses of vessel walls to chronically applied electrical stimuli.

8 mm long sections of common carotid arteries of conscious, freely moving rabbits were electrically stimulated with DC-impulses by chronically implanted gold-electrodes daily for 1/2 h or two times 1/2 h per day for 4--6 weeks. The electrodes were arranged in such a way that the wall was stimulated transmurally, In the initial phase the tension of the vessel wall increased. After 1/2 h of stimulation the artery wall relaxed at the stimulated section. Repetition of stimuli led to smooth muscle cell proliferation within the stimulated region. The smooth muscle cells formed a cushion protruding into the lumen of the artery mainly at the anodic site of the stimulation-arrangement. The proliferating cells were smaller than those of the underlying tunica media. They were identified as smooth muscle cells by electron-microscopy, contained relatively more lysosomes and endoplasmic reticulum than the original media-cells and produced collagen-fibrils and elastic fibrils as an extracellular matrix material. Below very thick cushions of smooth muscle cells necrosis developed. Animals which were additionally fed with 2% cholesterol in normal food developed typical atheromatous plaques at the site of the anodic stimuli.

Animals↗

PubMedDate not supplied

Response of cultured endothelial cells to mechanical stimulation.

Endothelial cells covering the luminal surface of vessels are exposed to at least two different mechanical forces: 1) fluid shear stress produced by the circulation of blood, and 2) periodic stretching and relaxing as a result of the diameter oscillations caused by blood pulsation. In this study we present an apparatus which was constructed to imitate the volume pulse with its typical incisura of the abdominal aorta. Using this apparatus, we exposed cultured endothelial cells to continuously produced cyclic and directional stretching and relaxation for three days. In all experiments cells remained attached and viable when subjected to mechanical stimulation. The vast majority of endothelial cells which underwent mechanical stimulation became elongated and oriented with their longer axis perpendicular to the direction of stretching (angle of cell orientation: alpha = 88.7 degrees +/- 12 degrees; means +/- SD), whereas cells on unstretched membranes had a cobblestone-like appearance and remained in random orientation. In the stretched cells, the factor of elongation was f = 6.8 +/- 1.3; means +/- SD; unstretched cells which exhibited a polygonal shape had a factor of elongation of f = 1.8 +/- 0.8; means +/- SD. In addition, the behavior of cytoskeletal components such as microfilaments and microtubules was examined in the process of cell orientation as both are actively involved in alterations of cell shape and cell migration. Actin filaments were oriented as both are actively involved in alterations of cell shape and cell migration. Actin filaments were oriented in parallel alignment perpendicular to the stretch direction (angle of actin filament orientation: beta = 90.4 degrees +/- 9 degrees; means +/- SD). A distinct orientation of microtubules was not observed, although a noticeable number of microtubules was observed to be in parallel alignment. Furthermore, microtubules of cells which underwent mechanical stimulation exhibited a pronounced asymmetric intracellular distribution with strongly fluorescent cytoplasmic areas in which microtubules seemed to be accumulated. The results indicate that endothelial cell elongation and orientation in vitro can be induced by periodic stretching and relaxation comparable to the periodic oscillations of the vessel wall due to blood pulsation in vivo.

Actin Cytoskeleton↗

PubMedDate not supplied

Selective percutaneous "biopsy" of atheromatous plaque tissue for cell culture.

The combination of percutaneous atherectomy and angioscopy enabled a selective "biopsy" of protruding atheromatous plaque material from 11 patients with arterial occlusive disease. The removed specimens were cultivated as adhering explants or single cells were obtained by enzymatic disintegration. The vast majority of the cultivated cells resembled fibroblasts, but could be identified as smooth muscle cells by their smooth muscle alpha-actin content. Proliferation rate was slow with 0.1 doublings per day. Endothelial cells were not observed by immunologic criteria. The described biopsy technique and in vitro evaluation of cultured human atheromatous plaque material may be useful for a better understanding of atherogenesis.

Actins↗

PubMedDate not supplied

Metabolic changes in modified smooth muscle cells of rabbit carotid arteries.

In the common carotid artery of rabbits, intimal myocyte proliferations were induced by daily repeated local electrical stimulation of the vessel wall in combination with a cholesterol containing diet given for 4 weeks. Some biochemical parameters of the morphologically modified intimal smooth muscle cells were studied and compared with those of samples obtained from nonstimulated tunica media of the contralateral carotid artery. The results show that in relation to dry weight both alkali extractable protein and DNA content of the proliferates are increased to about 125%. In the proliferates, the in-vivo tissue concentrations of glucose and glycogen are only 50-70% of the normal values, whereas the concentration of lactate is increased to about 160%. In-vitro incubation experiments of excised tissues samples from the intimal proliferates and normal media indicate that under an optimal supply of substrates the glucose uptake and lactate production of the proliferates are increased to 140% and 150%, respectively. This results provides evidence for an increased capacity of glycolysis in the proliferates, which in vivo may lead to a decrease in glucose concentration and to an increased concentration of lactate. This investigation shows that modified smooth muscle cells proliferating in the arterial intima exhibit an activated metabolism as seen in other models of arteriosclerosis.

Animals↗

PubMedDate not supplied

Significance of glucose for mechanical activity, flavin and pyridine nucleotide oxidation-reduction changes in isolated rat portal veins under ACh-stimulation.

The influence of extracellular glucose on isometric concentrations induced by acetylcholine was investigated under aerobic conditions in rat portal veins. In order to estimate the contractile potency of acetylcholine (ACh), dose-response curves of ACh and nor-epinephrine (NE) were compared. Simultaneously with the mechanical activity, changes in the redox states of pyridine nucleotides (PN) and flavoproteins were detected by a surface-fluorimetric method. Tissue concentrations of glucose, lactate, pyruvate, and ATP were determined parallel to ACh-induced contractions at corresponding intervals. In response to ACh-induced contractions, PN and flavoproteins were oxidized in vessels supplied with glucose. Following 6 h of glucose withdrawal, a small reduction or no change in the redox state was observed; ACh-induced contractions and tissue concentrations of glucose, lactate, pyruvate, and ATP were decreased. When glucose was added, the contraction force was restituted, and the tissue concentrations of glucose, lactate and PN were increased again. The ATP concentration remained decreased. The results suggest that isometric contractions of portal veins are significantly influenced by aerobic glycolysis. A direct effect of glucose on the excitability of vascular smooth muscle is discussed.

Acetylcholine↗

PubMedDate not supplied

Cell culture systems to study progression and inhibition of intimal proliferations.

Vascular smooth muscle cells, endothelial cells, and fibroblasts are the main cellular constituents of artery walls. Mass cultures and clone cultures of these cell types have meanwhile become valuable tools in the research of the genesis, pathophysiology, and therapy of vessel-wall diseases. With transfilter co-culture systems the three-layered construction of the artery wall can be imitated in vitro, and it has become possible to induce smooth muscle cell proliferates in these in vitro system which resemble, in many respects, intimal proliferates as they often occur after angioplasty, stent- or bypass operations in the form of secondary stenoses. With this technique the interaction of the three cell species of artery walls can be easily studied. The time-course of the development of smooth muscle cell proliferates in vitro resembles the in vivo scenario. Addition of oxidized lipoproteins and monocytes to the culture medium of transfilter cultures leads to atheroma-like proliferates. Culturing whole artery segments is another in vitro technique for induction of intimal proliferates, and enables the production of intimal proliferates in a way similar to transfilter culture systems. Because of the striking similarities of the cellular responses of transfilter- and organ-culture systems with in vivo processes in atherogenesis, and in the development of secondary stenoses after angioplasty, the described co-culture systems are suitable for studying the genesis, pathophysiology, and therapy of stenosing artery processes, as well as to obtain further insight into basic problems of cell interaction in vessel walls.

Animals↗

PubMedDate not supplied

Effect on intimal hyperplasia of dexamethasone released from coated metal stents compared with non-coated stents in canine femoral arteries.

PURPOSE: Polymer-coated, dexamethasone (DXM)-releasing stents were tested in order to assess the efficacy of DXM released locally for the prevention of stent restenosis due to intimal hyperplasia. METHODS: Strecker stents coated with a biodegradable membrane containing DXM were implanted percutaneously into the femoral artery in 14 dogs. The contralateral artery received a conventional non-coated stent serving as control. The drugs are eluted by degradation of the carrier membrane. Follow-up intraarterial digital subtraction angiography (DSA) was obtained at 3, 6, 9, 12, and 24 weeks with subsequent autopsy. Specimens for gross and microscopic pathology were obtained and histomorphometry was performed. RESULTS: Four of 14 DXM-coated stents showed thrombotic occlusion within the first 3 weeks; ten DXM-coated stents remained patent. At follow-up DSA, DXM-coated stents showed a significantly wider lumen than the non-coated stents. At morphometry there was less intimal hyperplasia over DXM-coated stents than over non-coated stents (p < 0.05). CONCLUSION: DXM-coated stents reduce neointimal hyperplasia in dogs when compared with non-coated stents.

Angiography↗