Characterization of prostacyclin synthesis in cultured human arterial smooth muscle cells, venous endothelial cells and skin fibroblasts.

The interaction of human platelets with one another and with the blood vessel wall is thought to be regulated in part by a balance between two arachidonic acid metabolites: thromboxane A₂, synthesized by platelets, and prostacyclin (PGI₂), synthesized by the vessel wall. We have studied the ability of cultured human vascular cells to synthesize PGI₂ from arachidonic acid. Four strains of human arterial smooth muscle cells synthesized a mean of 1.36 ng PGI₂ per 10⁵ cells, with a range of 0.2–5.3 ng PGI₂ per 10⁵ cells among the different strains. Human umbilical vein endothelial cells synthesized a mean of 7.16 ng PGI₂ per 10⁵ cells with a range of 2.3–14.0 ng per 10⁵ cells. In contrast, cultured human diploid skin fibroblasts synthesized only 0.27 ng PGI₂ per 10⁵ cells with a range of 0.05–0.6 ng per 10⁵ cells. When cultured cells were mixed with platelets, PGI₂ synthesis from added arachidonate was reduced rather than stimulated. Thus the major precursor cyclic endoperoxides utilized for PGI₂ synthesis are formed within the cells and not from endoperoxides synthesized by platelet cyclooxygenase. Aspirin has been proposed as an anti-thrombotic agent. Aspirin could be ineffective, however, if it inhibited not only platelet cyclooxygenase but that of vessel wall cells as well. Measurement of the rate constant or potency for aspirin inhibition of PGI₂ synthesis in cultured cells indicates that the cyclooxygenase in both cell types of the blood vessel wall is 14–44 fold less sensitive to aspirin inactivation than that in platelets, and appropriate levels of aspirin can selectively block human platelet thromboxane A₂ synthesis without compromising the capacity of the vasculature to produce PGI₂.     

Endogenous arachidonic acid metabolism by cultured arterial smooth muscle cells

Cultured aortic smooth muscle cells originated from healthy and atherosclerotic rabbits produce prostaglandins (namely prostacyclin) at a basal state. Prostaglandin secretion is dramatically reduced in atherosclerotic cells. This impairment was not correlated with any alteration of acyl hydrolase activities and probably involved a decrease of cyclooxygenase activities.     

Lipoprotein uptake by cultured human arterial smooth muscle cells.

Human arterial smooth muscle cells growing in tissue culture, in contrast to rat cells, preferentially bind and take up large, lipid-rich lipoproteins (125I-labeled low density and very low density lipoproteins) in comparison to the known difference in the propensity of these two species to develop atherosclerosis.     

Alpha-L-iduronidase activity in cultured skin fibroblasts and amniotic fluid cells

Using phenyl-α-l-iduronide as substrate, we have examined the level of α-l-iduronidase activity in homogenates of fibroblasts derived from normal individuals, from patients affected with α-l-iduronidase deficiency disorders (Hurler syndrome, Scheie syndrome, and a disease of intermediate severity presumed to be a Hurler/ Scheie compound) and from parents of such patients. Extracts derived from the affected individuals had no detectable α-l-iduronidase activity, whereas those derived from heterozygotes varied between 20% and 95% of the normal mean. Overlap between normal and heterozygous levels was reduced if the α-l-iduronidase activity was expressed on the basis of the β-galactosidase activity in the same homogenate. Cultured amniotic fluid cells from normal pregnancies had less than half as much α-l-iduronidase activity as fibroblasts from normal adults; this might cause problems in distinguishing a heterozygous fetus from an affected one by the enzyme assay alone.     

Regulation of low density lipoprotein receptor activity by cultured human arterial smooth muscle cells.

The ability of cultured human arterial smooth muscle cells to regulate low density lipoprotein (LDL) receptor activity was tested. In contrast to human skin fibroblasts incubated with lipoprotein deficient medium under identical conditions, smooth muscle cells showed significantly reduced enhancement of 125I-labeled LDL and 125I-labeled VLDL (very low density lipoprotein) binding. Smooth muscle cells also failed to suppress LDL receptor activity during incubation with either LDL or cholesterol added to the medium, while fibroblasts shoed an active regulatory response. Thus, in comparison with the brisk LDL receptor regulation characteristic of skin fibroblasts, arterial smooth muscle cells have and attenuated capacity to regulate their LDL receptor activity. These results may be relevant to the propensity of these cells to accumulate LDL and cholesterol and form "foam cells" in the arterial wall in vivo, a process associated with atherogenesis.     

Dimethyl sulfoxide induces microtubule formation in cultured arterial smooth muscle cells

Prolonged exposure of cultured arterial smooth muscle cells to 1% dimethyl sulfoxide (DMSO) resulted in a remarkable increase in cytoplasmic microtubules and an appearance of bundle-like aggregates of microtubules associated with ribosomes (microtubule-ribosome-complex). In this complex, fine filaments of 8 to 16 nm diameter were interspersed, some of which displayed continuity with the microtubules and were studded with a single array of ribosomes. The microtubule-ribosome-complex may represent an unusual aggregate of microtubules containing incompletely polymerized forms of newly synthesized tubulin induced by prolonged effect of DMSO.     

Laminin, fibronectin and type IV collagen in BM-like material from cultured arterial smooth muscle cells

1. The intra- and extracellular distribution of fibronectin and laminin was studied by immunofluorescence in cultures of rabbit and human arterial smooth muscle cells. 2. Basement membrane (BM)-like material was isolated from the cell layer of arterial smooth muscle cells cultures and analysed by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) and immunoblotting. The major 220-240 kD component of arterial BM-like material was identified as fibronectin. Also a 200 kD fibronectin band was observed. 3. The 200 kD subunit of laminin was contained in isolated BM-like material, but no slower migrating laminin chains were detected. 4. Collagens were prepared from pepsinized BM-like material. The band pattern as resolved by SDS-PAGE and silver staining suggested that type IV collagen is the major collagen of arterial BM-like material.     

Mineralocorticoid-induced increase in beta-adrenergic receptors of cultured rat arterial smooth muscle cells

We have investigated the effect of mineralocorticoids on beta-adrenergic receptors in cultured arterial smooth muscle cells. Mineralocorticoid (aldosterone) treatment resulted in a significant increase in beta-adrenergic receptors measured by [3H]dihydroalprenolol (DHA) binding. This effect required at least 20 hours of incubation with aldosterone and was completely blocked by cycloheximide (10 micrograms/ml), indicating protein synthesis was required for this response. Aldosterone at the concentration range of 10(-8)-10(-6) M increased [3H]DHA binding, but was ineffective at 10(-9) M. Scatchard analysis of [3H]DHA binding revealed that the observed significant increase in binding was due to an increased number of binding sites (P less than 0.05), and that the affinity was unchanged. The aldosterone (1 x 10(-8) M) effect was completely blocked by the combination of RU 38486 (10(-6) M) and spironolactone (10(-7) M), but not by the glucocorticoid antagonist RU 38486 alone. While basal c-AMP levels were not changed by aldosterone (10(-6) M) treatment, the isoproterenol (10(-6) M) stimulated level of c-AMP was significantly higher in cells treated with aldosterone (P less than 0.05). We conclude that aldosterone, acting through the mineralocorticoid receptor, has a direct effect on arterial smooth muscle cells mediated through modulation of beta-adrenergic receptors of these cells.     

Growth-mediated, density-dependent inhibition of endocytosis in cultured arterial smooth muscle cells

The effects of cell density and growth upon fluid phase endocytosis were investigated in quiescent and growing cultures of monkey arterial smooth muscle cells. Cells were maintained in a quiescent state of growth in 5% plasma-derived serum. Subsequent exposure of subconfluent cultures to the specific mitogens, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), or to whole blood serum, resulted in up to 4-fold increases in the rate of fluid endocytosis/cell. The changes began several hours after entry into G1 phase of the cell cycle and continued through S. The fraction of cells entering the growth cycle was variable (PDGF=FGF>EGF) and a close correlation existed between the rate of endocytosis and the fraction of [³H]thymidine-labelled cells (r = 0.929, p<0.01). At a range of cell densities, the rate of fluid endocytosis/cell was similar in sparse, confluent and post-confluent cultures of quiescent cells; in contrast, in growing cells there was density-dependent inhibition of endocytosis. Furthermore, when quiescent cells were in contact with each other and were then exposed to mitogens, the growth response was diminished and there was only a 25–50% increase in the rate of endocytosis, even in the presence of high concentrations of growth factors.These studies indicate that the influence of cell density upon fluid endocytosis in arterial smooth muscle cells is indirect in that it represents a secondary effect of decreased mitogenic response to specific growth factors.     

Orientation of cultured arterial smooth muscle cells growing on cyclically stretched substrates

Arterial smooth muscle cells from rabbit aortic media were grown in first subcultures on hydrophilized and collagen-coated silicone membranes which were then subjected to directional cyclic stretches and relaxations at a frequency of 50 times/min. The membranes were stretched 2, 5 and 10% beyond their resting length. Cells on unstretched and stationary membranes in the same chamber served as controls. The cells which were stretched with an amplitude of 2% remained in random orientation after 14 days of continuously performed cyclic stretching. The cells which were stretched 5% for 12 days orientated at an angle of 61 +/- 9 degrees to the direction of stretching, while the cells which were stretched with an amplitude of 10% for 6 days orientated at an angle of 76 +/- 5 degrees. The cells on the stationary and unstretched membranes remained in random orientation. We were able to confirm that the angle of orientation is reversible, i.e. preorientated cells changed their orientation during application of another stretching amplitude. The results suggest that stretching of the artery wall by blood pulsation may be a factor influencing the orientation of smooth muscle cells within the media of the artery wall and of those smooth muscle cells which proliferate into the subendothelial space after mechanical injury of the endothelium or electrical stimulation of the artery wall. An apparatus is presented which produces cyclic and directional mechanical stimuli similar to those which may occur in the artery wall.     

Carnitine transport in cultured muscle cells and skin fibroblasts from patients with primary systemic carnitine deficiency

Summary l-Carnitine transport was studied in cultured muscle cells and skin fibroblasts of patients with primary systemic carnitine deficiency and control subjects. In both cell culture types, two systems for carnitine transport were identified. The kinetic parameters for carnitine transport were remarkably similar in cultured muscle cells and skin fibroblasts. Normal rates and kinetic properties of carnitine transport were observed for both cell lines from patients with systemic carnitine deficiency. These studies do not rule out a defect in carnitine transport in vivo. This study was supported by research grants AM27451 and NS06277 from the National Institutes of Health and by a Research Center Grant from the Muscular Dystrophy Association.     

Two cholesterol ester hydrolases. Distribution in rat tissues and in cultured human fibroblasts and monkey arterial smooth muscle cells.

Hydrolytic activity against acetone-dispersed [4-14C]cholesterol oleate has been assayed as a function of pH in seven parenchymal tissues, blood cells, and plasma of the rat, as well as in cultured human fibroblasts and monkey (Macaca nemestrina) arterial smooth muscle cells. Both acid and neutral hydrolytic activities were present in all of these except rat plasma. The pH optima were in all cases close to pH 4.5 and pH 6.8. Acid activity was quite constant from tissue to tissue, while neutral activity varied greatly, being greatest in adrenal, testis, and adipose tissue. Subcellular fractionation of human fibroblasts allowed demonstration that activities at pH 4.5 and pH 6.8 were concentrated in different fractions, apparently lysosomal and polysomal, respectively. It appears most cell types, including fibroblasts and smooth muscle cells, contain two separate enzymes capable of hydrolyzing cholesterol esters. The neutral pH polysomal enzyme, which is especially prominent in certain tissues, may have a function related to the specialized roles of these tissues.     

Cell-associated proteoheparan sulfate from bovine arterial smooth muscle cells

Cell-associated proteoheparan sulfate has been isolated from bovine arterial smooth muscle cells preincubated with [35S]sulfate or a combination of [3H]glucosamine and [35S]methionine. The purified proteoheparan sulfate had an apparent Mr of 200,000 on calibrated Sepharose CL-2B columns. The glycosaminoglycan component (Mr approximately 30,000) was identified as heparan sulfate by its susceptibility to specific enzymatic and chemical degradation. After degradation of the proteoheparan sulfate by microbial heparitinase the resulting protein core had an apparent Mr of 92,000 on SDS-polyacrylamide gels. Its mobility was similar in the absence and presence of reducing agents indicating that the protein core consists of a single polypeptide chain. Pulse-chase experiments revealed that about 40% of the cell layer-associated proteoheparan sulfate was released into the medium, while the remainder was internalized and converted to smaller species through a series of degradation steps. Initially there was a proteolytical cleavage of the protein core generating glycosaminoglycan peptide intermediates with polysaccharides chains similar in size to the original. The half-life of the native proteoheparan sulfate was found to be about 4 h.