Gordon R. Campbell

Comparison of vascular smooth muscle cells from adult human, monkey and rabbit in primary culture and in subculture.

SummaryA method is presented for growing large numbers of pure isolated smooth muscle cells from adult human, monkey, and rabbit blood vessels in primary culture.In the first few days in culture these cells closely resembled those in vivo and could be induced to contract with angiotensin II, noradrenaline and mechanical stimulation. They stained intensely with antibodies against smooth muscle actin and myosin. Fibroblasts and endothelial cells did not stain with these antibodies thereby allowing the purity of each batch of cultures to be monitored. This was consistently found to be better than 99%. The smooth muscle cells modified or “dedifferentiated” after about 9 days in culture to morphologically resemble fibroblasts. At this stage cells could no longer be induced to contract and did not stain with the myosin antibodies. Intense proliferation of these cells soon resulted in a confluent monolayer being formed at which stage some differentiated characteristics returned. The modification or “dedifferentiation” process could be inhibited by the presence of a feeder layer of fibroblasts or endothelial cells, or the addition of cAMP to the culture medium.Smooth muscle cells which had migrated from explants in primary culture, and cells in subculture, had morphological and functional properties of “dedifferentiated” cells at all times.The advantages of differentiated rather than “dedifferentiated” smooth muscle cells in culture for the study of mitogenic agents in atherosclerosis is discussed.

What controls smooth muscle phenotype

Enzyme-dispersed smooth muscle cells from the adult pig aortic media in the first few days of primary culture are in the contractile phenotype and do not divide when challenged with 5% WBS. After 6--8 days the isolated cells spontaneously undergo a change in phenotype where contraction cannot be stimulated and the cells respond to mitogens in WBS by logarithmic growth. The change in phenotype is reversible if the cells are seeded sufficiently densely (5 x 10(4) to 1 x 10(5)/ml) that a confluent monolayer results after less than 1 week of proliferation, but is irreversible if the cells are seeded sparsely (1 x 10(3) to 5 x 10(3)/ml) and take more than 2 weeks of proliferation to reach confluence. When the cells are seeded so densely (10(6)/ml) that a confluent monolayer is present from day 1, the cells do not undergo a change in phenotype but remain indefinitely in the contractile state. The spontaneous modulation of phenotype of isolated smooth muscle cells can be inhibited by a confluent monolayer of contractile smooth muscle or endothelial cells in co-culture with the sparsely seeded smooth muscle such that the two cell layers are not in contact but bathed by the same nutrient medium. Smooth muscle modulation can also be inhibited by a factor extracted from pig and rabbit aortic tissue, and its effects mimicked by commercially available sodium heparin at 50 units/ml.

The Phenotypes of Smooth-Muscle Expressed in Human Atheroma

In 1866 Langhans described a population of subendothelial cells which he regarded as fibroblasts or fibrocytes. Since then, these cells have been described as modified smooth muscle cells (see REF. 2), multifunctional mesenchymal cells, differentiating or dedifferentiating smooth muscle cells,4 intermediate smooth muscle cells,5 or mesenchymal appearing.6 Ultrastructural studies indicated that these cells are smooth muscle of altered phenotypic and in recent years a wide variety of techniques has been used to demonstrate further structural and functional differences between medial smooth muscle cells and those in or around human atheromatous lesions (see TABLE I ) . Thus there is now overwhelming evidence that populations of smooth muscle cells in an atherosclerotic plaque (and involved in atherogenesis) express different phenotypes from those of the normal media. This article describes some of the different phenotypes expressed by smooth muscle cells in atheroma and discusses two experimental models in which many of these phenotypic populations can be reproduced.

Balloon catheter injury to rabbit carotid artery. I. Changes in smooth muscle phenotype.

Stereology was used to Investigate the changes In ultrastructure of smooth muscle cells during the formation of an experimental Intimal thickening Induced by Injury with an Inflated balloon catheter. The volume density of myofllaments in the cell cytoplasm was measured In smooth muscle cell-lined areas (which are freely permeable to Evans blue dye and, hence, stain blue) and In re-endothellalized areas (which remain white after Injection of Evans blue) of the rabbit carotid artery. Two weeks after Injury, the volume densities of myofllaments In the Intimal smooth muscle cells In both white and blue areas were significantly less than that for control medial smooth muscle (67.9%±3.6%; mean±SE), being 38.8%±1.0%and 35.9±3.3%, respectively. By 6 weeks after injury, the volume density had Increased significantly In both white (55.1%±3.4%) and blue areas (53.5%±3.0%), and these values did not change significantly by 18 weeks. The volume density of myofllaments in the luminal (lining) smooth muscle cells In the blue areas was significantly less than that of control medial cells and remained low (26.7%±2.1%) up to 18 weeks after Injury. The Initial balloon-Induced Injury caused considerable damage to the smooth muscle cells In the media, and the remaining medial cells underwent similar changes In ultrastructure to the cells in the neointlma. At 2 weeks, the cells had a low volume density of myofllaments (44.9%±2.4%), which returned to a level not significantly different from the control artery by 6 weeks after Injury. There were no differences in the estimates of the volume density of myofllaments between the Inner and outer media of the Injurled arteries. These findings suggest that, after Injury produced by a balloon catheter, the smooth muscle cells In both the media and the resultant Intimal thickening undergo a reversible change In ultrastructure.

Vasodilatation by acetylcholine is endothelium‐dependent: a study by sonomicrometry in canine femoral artery in vivo.

External diameter of the femoral artery was measured by sonomicrometry in the anaesthetized dog. Intra‐arterial acetylcholine lowered arterial pressure and thereby passively lowered diameter. When blood flow and distal resistance were controlled by roller pump and Starling resistor respectively, acetylcholine (0.1‐10 microM) and substance P (0.1‐1 nM) both caused up to 10% increase in diameter. Removal of endothelium by mechanically rubbing the artery lumen abolished the dilator response to acetylcholine and substance P but did not affect the response to nitroprusside. Constrictor responses to noradrenaline were unaltered by endothelium removal. Topical application of acetylcholine and substance P onto the adventitial surface of the artery also caused an increase in diameter but both agents were 50‐100 times less potent by this route compared with intra‐arterial infusion. These dilator responses were abolished by endothelium removal. In these circumstances acetylcholine caused constriction. We conclude that acetylcholine and substance P require an intact endothelium to elicit vasodilatation in vivo, at least for the large femoral artery. The results from the topical application experiments suggest that local neural release of vasoactive substances such as acetylcholine and substance P depend on an intact endothelium to cause vasodilatation.

Sympathetic ganglia in culture. I. Neurons.

Summary1. Phase-contrast microscopy, time-lapse cinematography, electron microscopy and fluorescence histochemistry were used to study neurons in cultured sympathetic ganglia of rat, guinea-pig and embryo chick.2. There was considerable variation in the morphology and size of neurons in all three species. Many neurons migrated into the outgrowth; those that migrated freely in rat and guinea-pig differed sufficiently from non-migratory neurons to allow a classification into type I (migratory) and type II (non-migratory). Type II neurons appeared to correspond to those seen in situ, but whether type I neurons represent immature or retarded neurons, or are due to culture conditions, is not clear. In the chick, no classification on this basis was possible; instead both migratory and non-migratory neurons showed a large and similar variation in nuclear size.3. In all three species both migratory and non-migratory neurons remained viable and noradrenaline was demonstrable histochemically for eight weeks (oldest cultures studied). Species differences were found in the extent of outgrowth of nerve fibers and accessory cells.4. Ultrastructural studies showed processes with features characteristic of sympathetic nerves in situ and of regenerating nerves. Profiles with other ultrastructural features were described and their identity discussed.5. The action of Nerve Growth Factor (NGF) at 1 unit/ml on sympathetic nerve fibers showed that guinea-pig was the most susceptible and chick the least. In the rat and guinea-pig, NGF increased levels of noradrenaline. NGF did not alter the appearance or relative numbers of type I and II neurons in rat and guinea-pig, but did affect the size distribution of both chick migratory and non-migratory neurons; some chick neurons were insensitive to NGF.

Somatostatin is contained in and released from cholinergic nerves in the heart of the toad bufo marinus

The heart of the toad Bufo marinus contained a substance with somatostatin-like immunoreactivity which eluted with somatostatin on reverse phase high pressure liquid chromatography. Immunoreactivity to somatostatin was localised histochemically to nerve fibers in muscle bundles of the sinus venosus, atria and ventricles and to nerve cell bodies in the sinus venosus and inter-atrial septum. Nerve cell bodies were localised both by interference contrast microscopy and immunohistochemistry; all detectable intracardiac neurons were immunoreactive. Synthetic somatostatin inhibited the rate and force of beat of atrial preparations, but did not affect the driven ventricle. Vagal stimulation caused inhibition of all cardiac chambers. After muscarinic blockade with hyoscine, vagal stimulation with 3 Hz or more still caused inhibition of the pacemaker and atrium, but not of the ventricle. The hyoscine-resistant vagal effects were diminished by about 60% after induction of tachyphylaxis to somatostatin. When when the vagus nerves were stimulated intermittently for 1 h at 10 Hz, in the presence or absence of hyoscine, the effect of somatostatin was reduced by about 60%. It is concluded that the cholinergic postganglionic neurons of the cardiac vagus contain somatostatin. When the vagus is stimulated at 3 Hz or more, the neurons release sufficient somatostatin to inhibit the pacemaker and atrial muscle.

Distinction between smooth muscle, fibroblasts and endothelial cells in culture by the use of fluoresceinated antibodies against smooth muscle actin

SummaryFITC-labelled antibodies against native actin from chicken gizzard smooth muscle (Gröschel-Stewart et al., 1976) have been used to stain cultures of guinea-pig vas deferens and taenia coli, rabbit thoracic aorta, rat ventricle and chick skeletal muscle. The I-band of myofibrils of cardiac muscle cells and skeletal muscle myotubes stains intensely. In isolated smooth muscle cells, the staining is located exclusively on long, straight, non-interrupted fibrils which almost fill the cell. Smooth muscle cells which have undergone morphological “dedifferentiation” to resemble fibroblasts with both phase-contrast microscopy and electronmicroscopy still stain intensely with the actin antibody. In those muscle cultures which contain some fibroblasts or endothelial cells, the non-muscle cells are not stained with the actin antibody even when the reactions are carried out at 37° C for 1 h or after glycerination. Prefusion skeletal muscle myoblasts also do not stain with this antibody.It is concluded that the actin antibody described in this report is directed against a particular sequence of amino acids in muscle actin which is not homologous with non-muscle actin. The usefulness of this antibody in determining the origin of cells in certain pathological conditions such as atherosclerosis is discussed.

Origin of myofibroblasts in the avascular capsule around free-floating intraperitoneal blood clots

Summary The origin of cells comprising the capsule which forms around free‐floating intraperitoneal blood clots has been examined in rats. One day after implantation the surface of the clot was covered by 1–2 layers of rounded nucleated cells. The majority of these cells were monocyte/macrophage in type with many cytoplasmic folds. Over the next 2 wk a thick capsule composed of dense connective tissue and myofibroblasts developed, completely covering the clot. The surface became covered by a contiguous layer of mesothelial cells, apparently derived from displaced cells of the damaged peritoneum. Detailed examination of the formation of the capsule suggested that a proportion of cells with the characteristic features of peritoneal macrophages gradually develop over a 3–4 d period the characteristic features of fibroblasts. These cells with time then develop filament bundles and a basal lamina to become myofibroblasts.

Lipid-Accumulation in Arterial Smooth-Muscle Cells - Influence of Phenotype

Isolated smooth muscle cells from the adult pig and rabbit aorta in primary culture undergo a spontaneous change in phenotype from a contractile to a synthetic state over 6-8 days, losing their capacity to contract and gaining the capacity to divide. The change in smooth muscle phenotype to the synthetic state is accompanied by distinct changes in the cells ability to metabolize LDL, with the rate of degradation of 125I-labelled LDL decreasing to about one fifth of the level in contractile state cells. This does not appear to be due to changes in the number or affinity of LDL receptors since saturable binding of LDL is unaltered. The specific activities of the lysosomal enzymes acid phosphatase and N-acetyl-beta-glucosaminidase increase with change to the synthetic state as do cytochrome c oxidase (mitochondria) and NADPH-dependent cytochrome c reductase (endoplasmic reticulum). In contrast there is a slight but not significant decrease in the specific activity of the lysosomal enzyme acid cholesteryl esterase of rabbit smooth muscle cells and a significant decrease in the activity of pig cells with change in phenotype to the synthetic state. Significantly more [3H]cholesteryl oleate is recovered in synthetic state than in contractile state cells following incubation with 20 micrograms/ml unlabelled LDL and [3H]sodium oleate. Morphologically there is no difference in the number of lipid droplets in contractile and synthetic state cells after incubation in 5% normolipemic serum, but in cells grown in 10% hyperlipemic serum for 4 days synthetic state cells become almost completely filled with lipid droplets while contractile state cells are unaffected. Lipid accumulation also occurs selectively in vivo in synthetic as compared with contractile state smooth muscle cells within intimal fibromuscular thickenings induced by de-endothelialization of the carotid artery of cholesterol-fed rabbits. We suggest that accumulation of lipid in smooth muscle cells of atherosclerotic plaques is related to reduced catabolism of LDL following smooth muscle phenotypic change from the contractile to synthetic state.