Rabbit Aortic Smooth Muscle Cells Research Articles

Effects of hypercholesterolemia on monokine-induced smooth muscle cell proliferation.

Macrophage/smooth muscle cell interactions play a role in atherogenesis and foreign body reactions to biomaterials. This study investigates the effect of a hypercholesterolemic diet on the ability of smooth muscle cells (SMCs) to respond to monokines which are produced in response to hypercholesterolemia, biomaterials or both. Peritoneal macrophages were harvested from rabbits fed either a normal (M phi NL) or a 2% cholesterol/6% peanut oil diet (M phi ATH) (plasma cholesterol 2840 vs 42.3 [p less than 0.005]). The macrophages were then cultured in the presence of either 1) polyglactin 910 (PG910), 2) Dacron, or 3) no biomaterial (control), and the media collected and pooled by week for the smooth muscle cell mitogenesis assays. Rabbit aortic smooth muscle cells were harvested and cultured from the same two groups of rabbits (SMCNL or SMCATH), quiesced in serum free media (48 h) followed by addition of the test media and 3H-TdR. The addition of either biomaterial to M phi NL-conditioned media increased 3H-TdR incorporation in both smooth muscle lines as compared to controls. PG910 resulted in significantly higher 3H-TdR incorporation than Dacron (weeks 3-5, p less than 0.005). The addition of either biomaterial to M phi ATH also increased 3H-TdR incorporation in both smooth muscle cell lines, however, the magnitude of the response was decreased as compared to the M phi NL-conditioned media in both cell lines (p less than 0.001 for either SMC line). In contrast to the M phi NL-conditioned media, the addition of Dacron to M phi ATH resulted in the highest level of 3H-TdR incorporation in both cell lines as compared to the media without biomaterial. The SMCNL had a higher response to both the monokines in conditioned media (2-fold) and to fetal bovine serum (3-fold) than the SMCATH (p less than 0.001). Although there is a generalized decrease in release of mitogens active on SMCs from M phi ATH, the M phi ATH exposed to Dacron release increased amounts of mitogenic factors, most active on the SMCATH cell line. A common mode of failure of small diameter Dacron grafts in man is pseudointimal hyperplasia, and it is inviting to postulate that the Dacron/macrophage/smooth muscle cell interactions in this atherosclerotic group of patients plays a role in the pathogenesis of this lesion.

Expression of calponin in rabbit and human aortic smooth muscle cells

Polyclonal antibodies to chicken gizzard calponin were used to localize calponin and determine calponin expression in rabbit and human aortic smooth muscle cells in culture. Calponin was localized on the microfilament bundles of cultured smooth muscle cells. Early in primary culture, calponin staining was accumulated preferentially in the central part of the cell body. With time in culture, the number of calponin-negative smooth muscle cells increased while the distribution of calponin in calponin-positive cells became more even along the stress fibers. Calponin content and the calponin/actin ratio decreased about 5-fold in rabbit aortic smooth muscle cells during the first week in primary culture and remained low in proliferating cells. The same tendency in calponin expression was observed when human vascular smooth muscle was studied. On cryostat sections of human umbilical cord, calponin antibodies mainly stained vessel walls of both the arteries and veins, although less intensive labelling was also observed in non-vascular tissue. When primary isolates of human aortic intimal and medial smooth muscle cells were compared with corresponding passaged cultures, it was found that calponin content was reduced about 9-fold in these cells in culture and was similar to the amount of calponin in endothelial cells and fibroblasts. Thus, high calponin expression may be used as an additional marker of vascular smooth muscle cell contractile phenotype.

Cholesterol efflux from cells to immunopurified subfractions of human high density lipoprotein: LP-AI and LP-AI/AII.

Using immunoaffinity chromatography, we separated human high density lipoprotein (HDL) into two subfractions: LP-AI, in which all particles contain apolipoprotein A-I (apoA-I) but no apoA-II, and LP-AI/AII, in which all particles contain both apoA-I and apoA-II. To compare LP-AI and LP-AI/AII as acceptors of cell cholesterol, the isolated subfractions were diluted to 50 micrograms phospholipid/ml, and then incubated with monolayer cultures of cells in which whole-cell and lysosomal cholesterol has been labeled with 14C and 3H, respectively. We used three cell types (Fu5AH rat hepatoma cells, normal human skin fibroblasts, and rabbit aortic smooth muscle cells). When these cells were prepared to contain normal physiological quantities of cholesterol (20-35 micrograms/mg protein), LP-AI and LP-AI/AII were nearly equally efficient in promoting efflux of both whole-cell and lysosomal cholesterol. For whole-cell cholesterol, the rate constants for efflux to LP-AI and LP-AI/AII were: 0.050/h and 0.053/h, respectively, with Fu5AH cells; 0.0063/h and 0.0074/h with GM3468 human skin fibroblasts; and 0.0076/h and 0.0079/h with rabbit aortic smooth muscle cells. When cholesterol in hepatoma cells or fibroblasts was elevated two- to threefold above normal, there was still not difference in efflux of whole-cell cholesterol to LP-AI and LP-AI/AII. In longterm incubations, the net depletion of cholesterol mass from cholesterol-enriched cells was either identical with the two HDL subfractions, or somewhat greater with LP-AI/AII.(ABSTRACT TRUNCATED AT 250 WORDS)

Rabbit aortic smooth muscle cell culture: A model for the pharmacological study of diabetesinduced alterations in cell proliferation

Atherosclerotic vascular disease is the most common complication of diabetes mellitus. Enhanced vascular smooth muscle cell proliferation plays a central role in atherosclerotic lesion formation. Studies using explant cultures have demonstrated that aortic smooth muscle cells from rats with experimental or genetic diabetes have enhanced rates of proliferation when compared to controls. However, this method of culture may select for cells with enhanced migratory potential. In the present studies, aortic smooth muscle cells were successfully cultured from control and diabetic rabbits after enzymatic and mechanical dispersion from thoracic aortic segments. The proliferative patterns of control cells were characterized and growth rates of diabetic cells were compared to controls. Primary cultures from control rabbits grew after an initial 5-day lag period to achieve threefold increases in cell number by 9 days. Subcultures of aortic smooth muscle cells entered the logarithmic phase of growth after 2 days, reaching the plateau phase of growth in 5–7 days and achieving three to fourfold increases in cell number. The final density to which cultures grew was not affected by the number of cells attached on day 1 for the range studied. Cells from diabetic rabbits displayed shorter doubling times and reached greater densities at confluence than did cells from controls. These data support the hypothesis that diabetes induces an atherogenic response. The dissociated rabbit aortic smooth muscle cell culture provides a model in which to study diabetes-induced modulation of cell proliferation that is amenable to pharmacological manipulation to investigate agonist and growth factor-induced responses.

Effects of oxidative modification of cholesterol in isolated low density lipoproteins on cultured smooth muscle cells

It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can participate in atherosclerosis. The present paper studied the effect of cholesterol oxidation in LDL on cultured vascular smooth muscle cells. LDL was oxidized by cholesterol oxidase (3-beta-hydroxy-steroid oxidase) which catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. Cholesterol oxidase treatment of LDL did not result in lipid peroxidation. Cultured rabbit aortic smooth muscle cells were morphologically changed following exposure to cholesterol oxidized LDL. Nile red, a hydrophobic probe which can selectively stain intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with oxidized or non-oxidized LDL cholesterol. LDL which did not undergo oxidation of its cholesterol had no effect on the cells. However, cellular nile red fluorescence intensity was increased as the pre-incubation time of cholesterol oxidase with LDL increased. This was supported by HPLC analysis which revealed that the oxidized cholesterol content of treated cells increased. These findings suggest that cholesterol oxidation of LDL can alter lipid deposition in the cells and change cell morphology. The oxidation of cholesterol in vivo may play an important role in the modification of LDL which could contribute to the generation of the lipid-laden foam cells.

Effects of cholesterol oxidase on cultured vascular smooth muscle cells

Cholesterol oxidase (3 beta-hydroxy-steroid oxidase) catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. The purpose of the present study was to investigate its effects on cultured vascular smooth muscle cells. Cultured rabbit aortic smooth muscle cells were morphologically altered after exposure to cholesterol oxidase in the presence of culture medium containing 10% fetal calf serum. If fetal calf serum was absent, cells were unaffected by the treatment. The extent of morphological change of the smooth muscle cells was dependent upon the time of exposure to the enzyme and the concentration of cholesterol oxidase employed. After moderate treatment with cholesterol oxidase, cells excluded trypan blue. Further, a specific mitochondrial marker DASPMI (dimethyl aminostyryl-methyl-pyridiniumiodine) which was used as a fluorescent index of cell viability, revealed that cell viability was unchanged after moderate cholesterol oxidase treatment. Nile red, a hydrophobic probe which selectively stains intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with cholesterol oxidase. Cellular nile red fluorescence intensity increased linearly with the time and concentration of cholesterol oxidase treatment. These results demonstrate that cholesterol oxidase alters lipid deposition in the cell and changes cell morphology. The primary site of action of cholesterol oxidase appears to be independent of the cell membrane itself and instead is dependent upon the lipid content in the surrounding culture media. These changes occur prior to the cytotoxic effects of extensive oxidation. Because oxidized cholesterol may play an important role in the pathogenesis of atherosclerosis, our results have implications for intracellular accumulation of lipids in smooth muscle cells during the atherosclerotic lesion.

The effects of an atherogenic diet on macrophage/biomaterial interactions

We previously reported that biomaterials differentially induce macrophages to secrete growth factors that mediate reendothelialization. The present study evaluates the effect of an atherogenic diet on macrophage/biomaterial interactions. Female New Zealand white rabbits were fed an atherogenic diet. Peritoneal macrophages were harvested from these as well as rabbits fed a normal diet and cultured in Minimum Essential Medium with platelet-poor serum. Dacron or polyglactin 910 were added to two of three conditions of both cell groups in passage 2. Conditioned media were collected weekly through week 15. Mitogenicity assays were performed with quiescent mouse embryonal (BALB/c3T3) fibroblasts, atherosclerotic rabbit aortic smooth muscle cells, and murine capillary lung (LE-II) endothelial cells. Mitogenic activity was assayed by scintillation counting of tritiated thymidine incorporation into deoxyribonucleic acid (DNA). Results showed increased mitogenic activity released by macrophages from atherosclerotic rabbits, in the absence of prosthetic material, when assayed against every cell line. In normal diet macrophages, polyglactin 910 stimulated mitogen release for every cell line, and Dacron yielded minimal mitogen release. In lipid diet macrophages polyglactin 910 slightly increased mitogen release for all three cell lines, whereas Dacron resulted in stimulation of DNA synthesis in smooth muscle cells and BALB/c3T3 cells but less DNA synthesis in LE-II cells than in control, no graft material, media. Western blotting demonstrated immunoreactivity to basic fibroblast growth factor in media from normal diet macrophages but only in the presence of polyglactin 910 or Dacron. Radioimmunoassay for platelet-derived growth factor B chain was negative in all groups, and polymerase chain reaction techniques to amplify transforming growth factor-beta messenger ribonucleic was negative. These data demonstrate the effect of in vivo dietary manipulation on macrophage activation as well as the effect of an atherogenic diet in modulating macrophage/biomaterial interactions. Additionally, different biomaterials differentially induce macrophages to release factors that stimulate and inhibit growth.

The effects of an atherogenic diet on macrophage/biomaterial interactions

We previously reported that biomaterials differentially induced macrophages to secrete growth factors that mediate reendothelialization. The present study evaluates the effect of an atherogenic diet on macrophage/biomaterial interactions. Female New Zealand white rabbits were fed an atherogenic diet. Peritoneal macrophages were harvested from these as well as rabbits fed a normal diet and cultured in Minimum Essential Medium with platelet-poor serum. Dacron or polyglactin 910 were added to two of three conditions of both cell groups in passage 2. Conditioned media were collected weekly through week 15. Mitogenicity assays were performed with quiescent mouse embryonal (BALB/c3T3) fibroblasts, atherosclerotic rabbit aortic smooth muscle cells, and murine capillary lung (LE-II) endothelial cells. Mitogenic activity was assayed by scintillation counting of tritiated thymidine incorporation into deoxyribonucleic acid (DNA). Results showed increased mitogenic activity released by macrophages from atherosclerotic rabbits, in the absence of prosthetic material, when assayed against every cell line. In normal diet macrophages, polyglactin 910 stimulated mitogen release for every cell line, and Dacron yielded minimal mitogen release. In lipid diet macrophages polyglactin 910 slightly increased mitogen release for all three cell lines, whereas Dacron resulted in stimulation of DNA synthesis in smooth muscle cells and BALB/c3T3 cells but less DNA synthesis in LE-II cells than in control, no graft material, media. Western blotting demonstrated immunoreactivity to basic fibroblast growth factor in media from normal diet macrophages but only in the presence of polyglactin 910 or Dacron. Radioimmunoassay for platelet-derived growth factor B chain was negative in all groups, and polymerase chain reaction techniques to amplify transforming growth factor-beta messenger ribonucleic was negative. These data demonstrate the effect of in vivo dietary manipulation on macrophage activation as well as the effect of an atherogenic diet in modulating macrophage/biomaterial interactions. Additionally, different biomaterials differentially induce macrophages to release factors that stimulate and inhibit growth.

Excess membrane cholesterol alters calcium movements, cytosolic calcium levels, and membrane fluidity in arterial smooth muscle cells.

The relations between membrane cholesterol content, basal (unstimulated) transmembrane 45Ca2+ movements, cytosolic calcium levels, and membrane fluidity were investigated in cultured rabbit aortic smooth muscle cells (SMCs) and isolated SMC plasma membrane microsomes. SMCs were enriched with unesterified (free) cholesterol (FC) for 18-24 hours with medium containing human low density lipoprotein and FC-rich phospholipid (PL) liposomes. This procedure increased cholesterol mass without affecting PL mass, resulting in an increase in the FC/PL molar ratio compared with controls in cells (67% FC increase, p less than 0.001; 43% FC/PL ratio increase, p less than 0.01) and in SMC microsomes (52% FC increase, p less than 0.05; 43% FC/PL ratio increase, p less than 0.05). Cholesterol enrichment also increased unstimulated 45Ca2+ influx (p less than 0.001) and efflux (p less than 0.05). Cellular cholesterol content correlated in a linear fashion with these changes (influx: r = 0.722, p less than 0.01; efflux: r = 0.951, p less than 0.05). In addition, cytosolic calcium levels increased approximately 34% (p less than 0.01) with cholesterol enrichment. The cholesterol-induced increase in 45Ca2+ influx was reversible with time and demonstrated sensitivity to the channel blockers. Fluorescence anisotropy measured from 5 degrees C to 40 degrees C using the fluorophore diphenylhexatriene showed decreased membrane fluidity in microsomal membranes obtained from cholesterol-enriched SMCs compared with controls (p less than 0.02). These results suggest that the SMC plasma membrane is very sensitive to cholesterol enrichment with liposomes or human low density lipoprotein and that increases in membrane cholesterol content increase cytosolic calcium levels in SMCs, are associated with a decrease in membrane fluidity, and unmask a new, or otherwise silent, dihydropyridine-sensitive calcium channel that may be involved in altered arterial wall properties with serum hypercholesterolemia.

Density-related expression of caldesmon and vinculin in cultured rabbit aortic smooth muscle cells

Quantitative immunoblotting techniques were used to study the effects of seeding density on the expression of caldesmon and vinculin variants, which are sensitive markers of vascular smooth muscle cell (SMC) phenotypic modulation in culture. Rabbit aortic SMC were seeded at different densities: 13 × 10 4 cells/cm 2 (high density), 3 × 10 4 cells/cm 2 (medium density), and 0.2 × 10 4 cells/cm 2 (low density) and cultured in the presence of 5% fetal calf serum. Irrespective of cell density and growth phase, caldesmon 150 was gradually and irreversibly substituted by caldesmon 77, but at high seeding density this substitution proceeded at a slower rate. The fraction of meta-vinculin (smooth muscle variant of vinculin) was reduced after seeding SMC in culture, but was reestablished when the cells reached confluency. Thus, high SMC seeding density is essential but not sufficient to keep vascular SMC cultured in the presence of serum in the contractile phenotype.

Effects of transforming growth factor- β1 on growth of aortic smooth muscle cells Influences of interaction with growth factors, cell state, cell phenotype, and cell cycle

Transforming growth factor (TGF)- β 1 may have different effects on cell proliferation depending on many conditions. This paper clarifies the effects of various conditions on the effect of TGF- β 1 on proliferation of cultured rabbit aortic smooth muscle cells (SMC) and also the time of its action during the cell cycle. TGF- β 1 at 10–10000 pg/ml inhibited DNA synthesis of SMC in the G 0 stage derived from normal media or atheromatous intima stimulated by either platelet-derived growth factor (PDGF), fibroblast growth factor, SMC-derived growth factor, or fetal bovine serum (FBS). TGF- β 1 also inhibited the growth of SMC in the growing state stimulated by either PDGF or FBS. TGF- β 1 was effective only when added to the culture within 2 h after stimulation of the G 0 state SMC with PDGF. It also inhibited increase in transcription of the c- myc protooncogene on stimulation of SMC with PDGF. These data suggest that TGF- β 1 inhibited proliferation of SMC irrespective of the cell phenotype, growth conditions, and growth factors present and that it exerted this inhibitory effect during the time of the G 0 G 1 transition.

Pituitary adenylate cyclase-activating polypeptide: a novel, long-lasting, endothelium-independent vasorelaxant

The vasoactivity of the 27- and 38-amino acid forms of the novel peptide pituitary adenylate cyclase-activating polypeptide (PACAP) was tested in vitro. Both forms of PACAP caused endothelium-independent vasodilation (assayed by their vasodilator action on rabbit aorta). When superfused for 1 min the relaxation EC 50 of PACAP27 was 23 ± 8 nM and of PACAP38 was 152 ± 66 nM. PACAP was 100-fold more potent than vasoactive intestinal polypeptide (VIP) (PACAP27 shows 68% amino acid sequence homology with VIP), and had a prolonged duration of action, a 1 min exposure to 1 μM PACAP27 lasting 135 ± 7 min and to 1 ft M PACAP38 108 ± 3 min. Adenylate cyclase activity in homogenates of rabbit aortic smooth muscle cells was increased by PACAP27 and PACAP38 with ECsoS of 4.4 and 0.73 nM, respectively. PACAP27 and PACAP38 are potent, long-lasting, endothelium-independent vasodilators.

Lysosomal hydrolysis of lipids in a cell culture model of smooth muscle foam cells

Rabbit aortic smooth muscle cells take up lipid droplets when they are presented using an inverted culture technique. These droplets were localized in secondary lysosomes as demonstrated by staining for acid phosphatase. Initially, 69% of the cell volume was occupied by lipid, and 94% of the lipid was in lysosomes. After a 24-hr clearance period, the cell volume occupied by lipid decreased to 53%, although there was no change in the fraction of cell lipid that was in lysosomes. To confirm that hydrolysis of droplet lipid was occurring in lysosomes, cultures were exposed to medium containing Sandoz 58-035, an inhibitor of acyl CoA:cholesterol acyl transferase, for 24 hr in the presence and absence of chloroquine, ammonium chloride, or methylamine. Although the hydrolysis of cholesteryl oleate was sensitive to these lysosomotropic agents, the hydrolysis of triolein was not. Using reconstituted LDL containing cholesteryl oleate and triolein, we demonstrated that the hydrolyses of cholesteryl oleate and triolein were equally sensitive to the lysosomotropic agents when the cells were not loaded with droplet lipid. However, in cells loaded with lipid, hydrolysis of LDL cholesteryl ester was sensitive to the lysosomotropic agents but hydrolysis of triolein was not. We therefore conclude that both droplet lipids were hydrolyzed in lysosomes, and we attribute the failure of the lysosomotropic agents to inhibit fully the hydrolysis of droplet triolein to the presence of a large mass of free fatty acids in the lysosome that maintains a sufficiently low pH to sustain the triglyceridase activity, but not the cholesteryl esterase activity, of the lysosomal acid lipase.

Oxidation of beta-very low density lipoprotein by endothelial cells enhances its metabolism by smooth muscle cells in culture.

We have previously shown that beta-very low density lipoprotein (beta-VLDL) incubated with bovine aortic endothelial cells (ECs) is bound and internalized more readily by cultured rabbit aortic smooth muscle cells (SMCs) than is beta-VLDL incubated in the absence of ECs, resulting in enhanced accumulation of cholesterol. To investigate the mechanism by which this occurs, beta-VLDL from hypercholesterolemic rabbit serum was incubated with cultured bovine aortic ECs. This resulted in the formation of thiobarbituric acid (TBA)-reactive material indicating extensive lipid peroxidation. The formation of TBA-reactive material, the increased metabolism of beta-VLDL by rabbit aortic SMCs, and the increased accumulation of cholesterol were prevented by superoxide dismutase, EDTA, several antioxidants, and, to a lesser extent, by 5,8,11,14-eicosatetraynoic acid, but not by acetylsalicylic acid, suggesting that potential oxidizing agents were the superoxide anion, metal ions, and lipoxygenase derivatives, but not cyclooxygenase derivatives. The percentage composition of phospholipid, protein, triglyceride, and free and esterified cholesterol of EC-modified beta-VLDL did not differ significantly from the unmodified lipoprotein. Displacement studies showed that only part of the interaction of both EC-beta-VLDL and unmodified beta-VLDL occurred through the B/E receptor and that the EC-beta-VLDL displaced 125I-beta-VLDL to a greater extent than did unmodified beta-VLDL. This indicated that the EC-beta-VLDL interacted more strongly with receptors on SMCs.

Does Nitric Oxide Inhibit Smooth Muscle Proliferation?

The lipid-soluble analogue 8-bromocyclic GMP did not inhibit thymidine incorporation into rabbit aortic smooth muscle cells initially in the contractile state but tended to inhibit incorporation into passaged cells by approximately 30%. In passaged cells, the nitrovasodilator drug SIN-1 (0.1-1 mM) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine produced a two- to fourfold increase in cyclic GMP concentration and an approximatively 50% reduction in [ 3 H]thymidine incorporation.

Modified low density lipoprotein isolated from atherosclerotic lesions does not cause lipid accumulation in aortic smooth muscle cells.

Foam cells in atherosclerotic lesions are derived not only from blood monocytes but also from smooth muscle cells (SMC). To better understand the mechanisms by which SMC may become lipid-laden, we have studied the catabolism by cultured rabbit aortic SMC of LDL derived from atherosclerotic lesions (A-LDL) previously shown to be chemically modified. A-LDL was isolated either from homogenates of atherosclerotic plaques in human aortas by affinity chromatography and gel filtration, or from nonhomogenized extracts of plaque minces by ultracentrifugation and gel filtration. Internalization of A-LDL by SMC or fibroblasts appeared to be mediated primarily via the LDL receptor since: 1) either unlabeled LDL or A-LDL could inhibit the degradation of 125I-labeled A-LDL or of 125I-labeled LDL, 2) the uptake of both A-LDL and LDL, as estimated by their abilities to stimulate cholesterol esterification, was reduced in cells in which LDL receptor expression was down-regulated; and 3) the uptake of both [3H]cholesteryl ether-labeled A-LDL and LDL by normal fibroblasts was significant and could be inhibited by excess LDL, but was negligible in receptor-negative fibroblasts. At saturating concentrations of lipoproteins, maximum cholesterol esterification in SMC was greater for LDL than for A-LDL. Over a 48-h incubation, A-LDL, like LDL, was unable to induce cellular cholesteryl ester accumulation. Cross-competition studies suggested that either the affinity of A-LDL for the LDL receptor was less than that of LDL, or that some particles in A-LDL are not internalized by SMC. The latter alternative was supported by the observations that some A-LDL particles had undergone aggregation, especially at high concentrations, and that aggregated forms of A-LDL or plasma LDL failed to be internalized and degraded by SMC. Collectively, these results are consistent with recognition of some of the A-LDL particles by the LDL receptor, but also suggest that, at least under in vitro conditions, A-LDL is unlikely to induce lipid accumulation in SMC resulting in SMC-derived foam cells.

Phenotypic expression of surface antigens of rabbit aortic smooth muscle cells in culture: Monoclonal antibody, 2P1A2, characteristic of smooth muscle cells present in atherosclerotic plaque, is not correlated with cell proliferation

The expression of smooth muscle cell (SMC) antigens was studied in culture by immunofluorescence and immunoelectron microscopy. As specific SMC markers, we used 2 monoclonal antibodies (MAb), 1PC1 and 2PIA2 which are able to detect atherosclerotic plaques in the rabbit. MAb 1PC1 recognizes an antigen expressed on the cell surface, starting on the 7th day in primary culture after serum activation, and then secreted. On a confluent SMC monolayers this antigen appears outside the cell as an important filamentous network. The kinetics of secretion of this external protein recognized by 1PC1 corresponds to the kinetics of the secretory phenotype described by Chamley-Campbell and Campbell ( Atherosclerosis, 40 (1981) 347). 2PIA2 MAb is specific for SMCs exclusively present in the rabbit atherosclerotic plaque. We studied the degree of reactivity of 2PIA2 with SMCs during primary cell culture. This “atherosclerotic” antigen of SMCs recognized by 2PIA2 is expressed in culture conditions by SMCs from rabbit normal media. This antigen appears after 3 days of serum activation, and heparin growth inhibition does not interfere with its expression. 2PIA2 recognized antigen is expressed during all cell cycle phases without amplification. 3 days after fetal calf serum (FCS) stimulation of cells which are in G 0 G 1 89% are labelled by 2P1A2, 4 days later G 0 G 1 positive cells constitute 49%. We conclude that 2P1A2 immunolabelling on the SMC surface reflects an activated state which is not correlated with SMC proliferation.

The 5'-flanking region of the mouse vascular smooth muscle alpha-actin gene contains evolutionarily conserved sequence motifs within a functional promoter.

The 5'-flanking, 5'-untranslated, and amino-terminal protein coding regions of the single-copy 13-kilobase mouse vascular smooth muscle (VSM) alpha-actin gene have been cloned and sequenced. Respectively, there is 73 and 89% homology from the start of transcription (+1) to a point 206 base pairs upstream when comparing mouse to chicken and mouse to human VSM alpha-actin 5'-flanking region sequences. Two proximal 16-base pair motifs containing putative cis-acting regulatory elements having the configuration CC(A/T)6GG were found to be 100% conserved and present in the same position upstream from the transcription start site in all three species. A third more distal CC(A/T)6GG-like motif was 100% conserved between only the mouse and human genes whereas a fourth motif was unique to the mouse gene. The two upstream motifs may be important in controlling VSM alpha-actin gene transcription in mammals. Cell transfection assays using hGH reporter gene fusion plasmids showed that all four CC(A/T)6GG elements were required for tissue-specific, core promoter activity and were able to direct hGH expression in both mouse BC3H1 myogenic cells and early-passage rabbit aortic smooth muscle cells. The core promoter was not active in mouse fibroblasts suggesting that the region between -372 and -143 may mediate tissue-restrictive expression of the VSM alpha-actin gene. A putative "cell density responsive element" may be located between -1074 and -372 since fusion plasmids containing this portion of the VSM alpha-actin 5'-flanking region were significantly more active in promoting hGH expression in inducible, density-activated BC3H1 myoblasts compared to aortic smooth muscle cells which are largely constitutive for VSM alpha-actin expression.

Alpha 1-adrenergic receptor mRNA level is regulated by norepinephrine in rabbit aortic smooth muscle cells.

Prolonged agonist exposure results in a decrease in the density of alpha 1-adrenergic receptors in rabbit aortic smooth muscle cells. A cDNA for the alpha 1-adrenergic receptor was used to assess the effect of norepinephrine on alpha 1-adrenergic receptor mRNA level in cultured vascular smooth muscle cells from the rabbit aorta. Norepinephrine caused a transient decrease (81% +/- 5%; n = 9) in alpha 1-adrenergic receptor mRNA. The effect was concentration dependent (EC50, approximately 0.3 microM; maximal effect, 10 microM). The maximum decrease occurred after 4 hr of exposure to norepinephrine and was followed by a gradual return to control levels by 24 hr. The decrease in mRNA level was blocked by prazosin, but not propranolol, and was mimicked by phenylephrine. These results indicate that the effect is mediated by stimulation of the alpha 1-adrenergic receptor and suggest that it involves one or more alpha 1-adrenergic-coupled second messenger pathways. The decrease in alpha 1-adrenergic receptor mRNA caused by norepinephrine exceeds that caused by actinomycin D, suggesting that norepinephrine may cause a decrease in the stability of alpha 1-adrenergic receptor mRNA. Actinomycin D also blocked the norepinephrine-induced decrease in mRNA level, further suggesting that the effect of norepinephrine requires induction of transcription, presumably leading to synthesis of a labile factor that is necessary for the effect of norepinephrine on alpha 1-adrenergic receptor mRNA level.

Concanavalin A‐ and fetal‐calf‐serum‐induced rounding and myosin light chain phosphorylation in cultured smooth muscle cells

Rabbit aorta smooth muscle cells (SMC) in long-term culture retracted in less than 10 min in response to a sequential order of stimulations by concanavalin A (Con A) and fetal calf serum (FCS). With additional continuous stimulation by FCS, the SMC took on a circular shape and were anchored to the substrate by retraction fibrils. This rounding was observed only when the cells were sequentially stimulated by Con A and FCS. Depletion of intracellular Ca2+ stores by the addition of EGTA and Ca2+ ionophores inhibited the rounding. Transient phosphorylation of MLC20 was observed in the initial stage during the SMC rounding. The extent of monophosphorylated MLC20 increased for up to 5 min to a maximal value of 49%. The diphosphorylated form reached a maximal value of 29% within 2 min; then both forms of MLC20 decreased. The process of the SMC rounding was inhibited by antimycin A or cytochalasins, in a dose-dependent manner, findings which suggested a dependency on both metabolic energy and actin-containing microfilaments. The smooth-muscle-relaxing agent, HA1077, also inhibited the process of SMC rounding. These observations suggest that a cellular contractile process might be involved in rounding of SMC.