Immunoreactive forms of caldesmon in cultivated human vascular smooth muscle cells

150 kDa caldesmon was shown to be characteristic of vascular smooth muscle cells in normal tissue rather than in subculture. Subcultured smooth muscle cells from human aorta contained only the 70 kDa immunoreactive form of caldesmon. During the course of primary culture the amount of 150 kDa caldesmon as well as metavinculin decreased significantly whilst 70 kDa caldesmon became the predominant form, and by the onset of cell division the 150 kDa form was practically substituted by 70 kDa caldesmon. The data show that the predominance of 150 kDa caldesmon is characteristic of contractile smooth muscle cells, while in proliferating cells 70 kDa caldesmon is expressed.     

Expression of meta-vinculin associated with differentiation of chicken embryonal muscle cells

We confirmed the existence of meta-vinculin, which cross-reacts immunologically with vinculin and has a slightly higher molecular weight than the latter. The immunological cross-reactivity between meta-vinculin and vinculin was confirmed with monoclonal antibodies against vinculin. Furthermore, we found that this protein is present only in either smooth or striated muscle, but is absent in non-muscular tissues and that the expression of this protein is associated with a differentiation of muscle cells either in vivo or in vitro. Microinjection experiments of fluorescently labelled vinculin and/or meta-vinculin into the cytoplasm of cultured myotubes suggested that meta-vinculin may be localized at sites similar to vinculin in muscle cells.     

Immunolocalization of meta-vinculin in human smooth and cardiac muscles

Meta-vinculin, a vinculin-related protein, has been isolated from human uterus smooth muscle. Specific antibodies to meta-vinculin, which distinguish between meta-vinculin and vinculin, were prepared by absorption of anti-meta-vinculin serum on vinculin coupled to nitrocellulose. Meta-vinculin specific antibody demonstrates only smooth and cardiac muscle specificity and is able to cross-react with a small 21-kD fragment of the meta-vinculin polypeptide chain. This antibody does not interact with protease resistant 95-kD core shared by vinculin and meta-vinculin. Meta-vinculin specific antibody was used for the localization of meta-vinculin in smooth and cardiac muscles by the indirect immunofluorescence method. At the light microscopy resolution level it was found that meta-vinculin and vinculin are localized in the same cellular adhesive structures. Meta-vinculin is present in membrane-associated microfilament-bound plaques of smooth muscle, in intercalated discs and costameres of cardiac muscle. In primary culture of smooth muscle cells from human aorta, meta-vinculin and vinculin were found to be present in focal contacts of the cells. During the cultivation of smooth muscle cells, the quantity of meta-vinculin decreased progressively and finally meta-vinculin completely disappeared from the focal contacts. The data show that in smooth and cardiac muscles meta-vinculin could be a structural component of microfilament-membrane attachment sites, defined earlier by the localization of vinculin.     

Expression of fibronectin variants in vascular and visceral smooth muscle cells in development

Monoclonal antibodies recognizing extra domain A (ED-A) and extra domain B (ED-B) fibronectin (FN) sequences were used to characterize FN variants expressed in human vascular smooth muscle cells (SMC) during fetal and postnatal development and to compare spectrum of FN variants produced by vascular and visceral SMC. In 8- to 12-week-old fetuses both ED-A-containing FN (A-FN) and ED-B-containing FN (B-FN) were found in all smooth muscles studied--aorta, esophagus, stomach, and jejunum. By 20-25 weeks of gestation relative amounts of both A-FN and B-FN were reduced significantly in the aortic media (fivefold for A-FN and twofold for B-FN), while in visceral SMC only B-FN content was decreased. All the adult visceral smooth muscles examined contained A-FN rather than B-FN. Therefore, the cells from adult aortic media appear to be the only SMC so far known to produce FN that contains neither ED-A nor ED-B. Moreover, the data obtained show that, unlike other cells, medial SMC are embedded in vivo in the extracellular matrix that contains FN lacking both ED-A and ED-B. SMC from the minor intimal thickenings in the human child aorta as well as those from the atherosclerotic plaques produce A-FN rather than B-FN. We conclude that (1) vascular SMC change the spectrum of produced FN variants at least twice--during prenatal development between 12 and 20 weeks of gestation, and during the postnatal period, when they are recruited into the intimal cell population; (2) the production of FN variants in visceral SMC is also developmentally regulated; (3) all visceral SMC unlike the cells from adult aortic media produce A-FN; (4) the presence of ED-A and ED-B sequences in the FN molecule is not necessary for the extracellular matrix assembly in vivo.     

Polymorphism of smooth muscle cells in atheromatous plaques of the human aorta

The expression of cell cytoskeleton proteins in atheromatous plaques of human aorta was investigated using double immunofluorescence technique and a set of antibodies. It was found that in 4 out of 12 plaques some smooth muscle cells (SMC) were stained by monoclonal antibodies to desmin. No such cells were detected in apparently unaffected aortic intima. In addition to typical SMC and these cells, the cells unstained by antisera to smooth muscle myosin but reacting with monoclonal antibodies to vimentin and SMC surface were revealed in all plaques adjacent to the central fatty mass.     

Expression of receptors for human angiogenin in vascular smooth muscle cells.

Human angiogenin is a plasma protein with angiogenic and ribonucleolytic activities. Angiogenin inhibited both DNA replication and proliferation of aortic smooth muscle cells. Binding of 125I-angiogenin to bovine aortic smooth muscle cells at 4 degrees C was specific, saturable, reversible and involved two families of interactions. High-affinity binding sites with an apparent dissociation constant of 0.2 nm bound 1 x 104 molecules per cell grown at a density of 3 x 104.cm-2. Low-affinity binding sites with an apparent dissociation constant of 0.1 micrometer bound 4 x 106 molecules.cell-1. High-affinity binding sites decreased as cell density increased and were not detected at confluence. 125I-angiogenin bound specifically to cells routinely grown in serum-free conditions, indicating that the angiogenin-binding components were cell-derived. Affinity labelling of sparse bovine smooth muscle cells yielded seven major specific complexes of 45, 52, 70, 87, 98, 210 and 250-260 kDa. The same pattern was obtained with human cells. Potential modulators of angiogenesis such as protamine, heparin and the placental ribonuclease inhibitor competed for angiogenin binding to the cells. Together these data suggest that cultured bovine and human aortic smooth muscle cells express specific receptors for human angiogenin.     

Monoclonal antibodies that distinguish between human aorta smooth muscle and endothelial cells

A monoclonal antibody has been generated that interacts with the surface of cultured human aorta smooth muscle cells and does not bind to the endothelial cells from aorta and umbilical vein. An antigen recognized by the antibody has a molecular mass of 330 kDa as determined by electrophoresis of immunoprecipitate in SDS-polyacrylamide gel. The same antigen appeared to be present on the fibroblast surface while neither immunofluorescence, flow cytofluorimetry nor immunoprecipitation reveal it on the endothelial cell surface or in the Triton X-100 extract.     

Expression of calponin in rabbit and human aortic smooth muscle cells

Summary 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,ccalponin 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.     

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 x 10(4) cells/cm2 (high density), 3 x 10(4) cells/cm2 (medium density), and 0.2 x 10(4) cells/cm2 (low density) and cultured in the presence of 5% fetal calf serum. Irrespective of cell density and growth phase, caldesmon150 was gradually and irreversibly substituted by caldesmon77, 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.     

Calcium channels of amphibian stomach and mammalian aorta smooth muscle cells.

Whole-cell and single-channel calcium currents were studied using single smooth muscle cells enzymatically-isolated from stomach of Amphiuma tridactylum and from guinea-pig aorta. These cells have a high specific resistance and can sustain calcium action potentials after suppression of potassium currents. Dialyzed Amphiuma smooth muscle cells had calcium currents which were stable for several hours whereas the calcium currents of aortic cells ran down quickly. Single channel calcium currents in cell-attached patches behaved similarly for the two cell types. Calcium channel conductance in 110 mM barium was 12 pS and the mean open time was 1.4 ms at a nominal membrane potential of +10 mV. Exposure of both cell types to BAY K8644 resulted in a dramatic prolongation of the calcium channel open times and a shift in the probability of opening to more negative potentials. Low-threshold calcium channels were not identified in the extensively studied amphibian cells. High-threshold calcium channels therefore appear to be the primary pathway for the calcium influx that produces contraction in these smooth muscle cells.     

Platelet-derived growth factor-induced alterations in vinculin distribution in porcine vascular smooth muscle cells

Exposure of porcine vascular smooth muscle cells to platelet-derived growth factor (PDGF; 18-180 ng/ml) but not epidermal growth factor (EGF; 30 ng/ml), somatomedin C (SmC; 30 ng/ml), or insulin (10 microM), results in a rapid, reversible, time- and concentration-dependent disappearance of vinculin staining in adhesion plaques; actin-containing stress fibers also become disrupted following exposure of cells to PDGF. Disappearance of vinculin staining from adhesion plaques is also caused by 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 200-400 nM), though the time course of the disappearance of vinculin staining under these conditions takes longer than in cells exposed to PDGF. The PDGF-induced removal of vinculin from adhesion plaques was inhibited in a concentration-dependent fashion by 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8; 0.25-4 microM) and leupepetin (2-300 microM), and by n-alpha-tosyl-L-lysine chloromethylketone (TLCK; 100 microM) and trifluoperazine (TFP; 2.5 microM). Addition of PDGF to vascular smooth muscle cells caused a rapid, transient increase in cytosolic free calcium, from a basal resting level of 146 +/- 6.9 nM (SEM, n = 62) to 414 +/- 34 nM (SEM, n = 22) as determined using the calcium-sensitive indicator Fura-2 and Digitized Video Microscopy. This increase in cellular calcium preceded the disappearance of vinculin from adhesion plaques and was partially blocked by pretreatment of cells with TMB-8 but not leupeptin. This rise in cytosolic free calcium was found to occur in approximately 80% of the sample population and displayed both spatial and temporal subcellular heterogeneity. Exposure of cells to TPA (100 nM) did not result in a change in cytosolic free calcium. Both PDGF (20 ng/ml) and TPA (100 nM) caused cytosolic alkalinization which occurred after PDGF-induced disruption of vinculin from adhesion plaques, as determined using the pH-sensitive indicator BCECF and Digitized Video Microscopy. PDGF stimulated DNA synthesis and vinculin disruption in a similar dose-dependent fashion. Both could be inhibited by leupeptin or TMB-8. These results suggest that 1) exposure of vascular smooth muscle cells to PDGF is associated with the disruption of vinculin from adhesion plaques, 2) PDGF-induced vinculin disruption is regulated by an increase in cytosolic calcium (but not cytosolic alkalinization), and involves proteolysis; 3) activation of protein kinase C also causes vinculin removal from adhesion plaques but by a calcium-independent mechanism, and 4) the cellular response to PDGF-stimulated increases in cytosolic free calcium is heterogeneous.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heparin induces specific protein release from human intestinal smooth muscle cells

Human intestinal smooth muscle cells have recently been identified as the major cell type responsible for stricture formation in Crohn's disease. Heparin, a sulfated glycosaminoglycan, has been shown to be a key modulator of vascular smooth muscle cell growth both in vivo and in vitro and to affect the release of proteins from these cells. Heparin has also been shown to affect the growth of human intestinal smooth muscle cells. In this report we demonstrate that heparin, in addition to its effects on proliferation, also has very specific effects on proteins released by these cells in vitro. Examination of the culture medium proteins of heparin-treated human intestinal cells revealed an increase in three proteins of molecular weight between 150-250 kd, an increase in a 37 kd protein and a decrease in synthesis of lower molecular weight (less than 20 kd) proteins. In substrate-attached material a transient effect on a 48 kd protein was observed. No effects on intracellular labeled proteins could be demonstrated. The 35S-methionine labeled protein profile of human intestinal smooth muscle cells exposed to heparin is similar to that observed in rat vascular smooth muscle cells yet distinct differences do exist. Extracellular processing does not account for the released proteins nor is de novo protein synthesis required suggesting that altered intracellular protein processing is the mechanism for the heparin-induced protein pattern. The release of specific proteins following exposure to heparin may reflect a significant influence of this glycosaminoglycan on the metabolism of smooth muscle cells in general and particularly in the human intestine.     

Characterization of the collagen synthesized by cultured human smooth muscle cells from fetal and adult aorta.

Smooth muscle cells were grown from explants of the tunica media of fetal and adult human aorta. Collagen was isolated after incubation with [14C]glycine and was characterized by ion-exchange chromatography. All cells investigated synthesized two types of collagen: Type I (chain composition [alpha1(I)]2alpha2) and type III (chain composition [alpha1(III)]3). The collagen made by cells from adult donors contained approximately 70% type I and 30% type III collagen. This corresponds to the collagen composition in teh original tissue. No age-relate change in the type I/type III ratio was found with cells from donors between 9 and 67 years of age. On the other hand, the type III portion of the collagen made by fetal cells was markedly less (about 15-20% of total collagen).     

Expression of TRPC homologs in endothelial cells and smooth muscle layers of human arteries

TRPC channels are a group of Ca²⁺-permeable nonselective cation channels that mediate store-operated and/or agonist-stimulated Ca²⁺ influx in a variety of cell types. In this study, we extensively examined the expression patterns of TRPC homologs in human vascular tissues. RT-PCR amplified cDNA fragments of TRPC1 (505 bp), TRPC3 (372 bp), TRPC4 (499 bp), TRPC5 (325 bp), TRPC6 (509 bp), and TRPC7 (187 bp) from RNA isolated from cultured human coronary artery endothelial cells. In situ hybridization yielded strong labeling of TRPC1,3–6 in the endothelial and smooth muscle cells of human coronary and cerebral arteries. TRPC7 labeling was exclusively found in endothelial cells but not in smooth muscle cells. Results from immunohistochemical staining were consistent with those from in situ hybridization. Similar expression patterns of TRPC homologs were also observed in arterioles and vaso vasora. In conclusion, our study indicates that TRPC homologs are widely expressed in human vessels of all calibers, including medium-sized coronary arteries and cerebral arteries, smaller-sized resistance arteries, and vaso vasora. These results suggest a ubiquitous role of TRPC homologs in regulating blood supply to different regions and in controlling arterial blood pressure.     

Cyclic strain induces expression of specific smooth muscle cell markers in human endothelial cells

The objective of this study was to determine whether cyclic strain could promote human umbilical vein endothelial cells (HUVECs) to express markers in common with the mature smooth muscle cell (SMC) phenotype, suggesting endothelial cell to SMC transdifferentiation. HUVECs were cultured on stretched membranes at 10% stretch and 60 cycles/min for 24-96 hr, and demonstrated elongation with enhanced and organized F-actin distribution. By using real-time polymerase chain reaction analysis, the mRNA levels of five specific SMC markers, SM22-alpha, alpha-smooth muscle actin (alpha-SMA), caldesmon-1, smooth muscle myosin heavy chain (SMMHC), and calponin-1 were significantly increased in cyclic strain-treated HUVECs as compared with those in static control cells. Protein levels of SM22-alpha and alpha-SMA were also substantially increased by Western blot and immunofluorescence staining. In addition, two specific endothelial markers, von Willebrand factor (vWF) and vascular endothelial growth factor receptor-2 (VEGFR-2), showed a reduction in mRNA expression. In addition, cyclic strain-induced increase of SM22-alpha and alpha-SMA expression were reversible when cells were cultured back to the static condition. These results demonstrate a possible endothelial cell to SMC transdifferentiation in response to cyclic strain. Hemodynamic forces in modulating endothelial phenotype may play an important role in the vascular system.