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.     

Diversity of vinculin/meta-vinculin in human tissues and cultivated cells. Expression of muscle specific variants of vinculin in human aorta smooth muscle cells

Microheterogeneity of different vinculin and meta-vinculin isoforms in adult human tissues and cultured cells was studied by two-dimensional gel electrophoresis and immunoblotting technique. Four isoforms of vinculin (alpha, alpha', beta, and gamma) and two isoforms of meta-vinculin (alpha and beta) were resolved. alpha-, alpha'-, and beta-isoforms of vinculin were found in all cell types and tissue samples analyzed in the present study. gamma-Isoform of vinculin and both alpha- and beta-isoforms of meta-vinculin were found in smooth (aorta wall and myometrium) and cardiac muscle, rather than in skeletal muscle, liver, foreskin fibroblasts, and macrophages. In the primary culture of human aorta smooth muscle cells, the fractional content of gamma-isoform of vinculin and meta-vinculin was dramatically reduced, and, by the onset of intensive cell division, the proteins could hardly be detected. Subcultured human aorta smooth muscle cells did not contain gamma-vinculin and meta-vinculin. We analyzed the microheterogeneity of vinculin and meta-vinculin in three smooth muscle layers of human aorta wall--media, muscular-elastic (adjacent to media) intima, and subendothelial (juxtaluminal) intima. It was shown that in media the fractional content of gamma-isoform of vinculin was 45% and meta-vinculin, 42%; in muscular-elastic intima the fractional content of gamma-vinculin was 42% and meta-vinculin, 36%. However, in subendothelial intima, the share of these proteins was significantly lower than in adjacent muscular-elastic intima and media. Isoactin pattern that is characteristic of smooth muscle was identical in all aortic layers, thus proving the smooth muscle origin of subendothelial intima cells. These findings demonstrate that human aortic smooth muscle cells in vivo and in vitro undergo coordinated differential expression of smooth muscle specific variants of vinculin, i.e. gamma-vinculin and meta-vinculin.     

Developmental changes in expression of contractile and cytoskeletal proteins in human aortic smooth muscle

To describe phenotypic changes of human aortic smooth muscle cells (SMCs), proportion of smooth muscle and nonmuscle variants of actin, myosin heavy chains (MHCs), vinculin, and caldesmon, during prenatal and several months of postnatal development was determined. In aortic SMCs from 9-10-week-old fetus, both nonmuscle and smooth muscle-specific variants of all four proteins were present, however, the nonmuscle forms were more abundant. During development, a shift towards the expression of muscle-specific variants was observed, although the time course of changes in protein variant content was not similar for all the proteins studied. By the 24th week of gestation, fractional content of alpha-smooth muscle actin and smooth muscle MHCs was rather close to that in the mature SMCs, and comprised approximately 80 and 90%, respectively, of the levels characteristic of SMCs from adult aortic media. On the contrary, fractional ratio of meta-vinculin and 150-kDa caldesmon was still rather low in the aorta from the 24-week-old fetus, did not increase in a 2-month-old child aorta, and did not reach the level characteristic of mature SMCs even in the 6-month-old child aorta. Thus changes in alpha-smooth muscle actin and smooth muscle MHC fractional content occur mainly during the prenatal period of development, before the 24th week of gestation; while meta-vinculin and the 150-kDa caldesmon proportion increases mainly in the postnatal period, during several months after birth. In the "Discussion," phenotypes of SMCs from developing aorta were compared to those from different layers of the adult aortic wall.     

Properties of smooth muscle meta-vinculin

Quantitative studies show that meta-vinculin is ninefold more soluble in 0.6 M salt than in the 0.01 M salt buffers used to extract vinculin. Based on this finding, we have developed a protocol for the purification of meta-vinculin in 43% yield and 98% purity from a high salt extract of gizzard smooth muscle. In contrast to our earlier extraction studies, which were done on unfixed cryostat sections (30), the present studies done on tissue homogenates show that nonionic detergents are not required for solubilization of meta-vinculin. Furthermore, neither purified nor partially purified meta-vinculin binds to Triton X-114 micelles. Purified meta-vinculin is a monomeric, asymmetric molecule with a Stokes radius of 50.9 A, a sedimentation coefficient of 6.35S, and a frictional ratio of 1.46. The calculated molecular weight of meta-vinculin is 145,000. Meta-vinculin has two isoforms of pI 5.9 and 6.2, and is phosphorylated in vivo to eightfold greater specific activity than vinculin. On immunoblots of smooth muscle proteins, [125I]meta-vinculin binds specifically to talin and also to unidentified polypeptides of 180, 150, 95, 70, 68, and 45 kD. On two-dimensional peptide maps, iodinated vinculin and meta-vinculin have at least 95% of their major chymotryptic peptides in common, but each protein also has at least one highly labeled peptide that appears to be unique. Comparative peptide maps of high salt soluble meta-vinculin and the low salt soluble 152-kD protein (described by Feramisco, J.R., J.E. Smart, K. Burridge, D. Helfman, and G.P. Thomas, 1982, J. Biol. Chem., 257:11024-11031) demonstrate extensive similarities among the vinculin-like proteins but suggest a lack of complete identity. In vivo pulse-chase experiments show that meta-vinculin and vinculin do not have a precursor-product relationship. The biochemical and structural differences found between vinculin and meta-vinculin suggest that there is a unique function for meta-vinculin in smooth muscle.     

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.     

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.     

Regional functional heterogeneity of aortic smooth muscle cells in rats

The aorta is a magistral artery, which has been traditionally looked upon as a vessel whose properties are invariable throughout its length. However, in the most recent decade, there have been accumulated data that provide evidence that different aorta sections arise from different embryonic origins and that the population of smooth muscle cells making up the vessel’s wall is, consequently, heterogenic. Tracing the fate of smooth muscle cells, the basic components of the vessel, with the aid of genetic marking methods revealed that the cells’ response to various factors is largely determined by the embryonic origin of a certain cell population. However, functional differences between the smooth muscle cells making up different aorta sections remain poorly understood. The aim of the current work was to compare the functional characteristics of the populations of aortic wall smooth muscle cells obtained from the aorta sections differing by their embryonic origin. Towards this end, we obtained smooth muscle cell cultures from the three aorta sections of linear rats, namely, the neural crest derived ascending thoracic aorta, the somites derived descending thoracic aorta, and splanchnic mesoderm derived abdominal aorta. Using immunocytochemistry and Western blotting, the cells from the different regions of aorta were compared on the basis of smooth muscle actin, vimentin, and SM22 content in them. Cell proliferation rate was estimated using the growth curves method. We have demonstrated that the three smooth muscle cell populations arising from different embryonic origins differ in their morphological characteristics as well as by smooth muscle actin and SM22 content. We have shown that smooth muscle cells from the ascending aorta proliferate more actively than the corresponding cells from the descending thoracic aorta. Thus, the functional properties of the populations of rat aortic smooth muscle cells are different and depend on the embryonic origin of the aorta section from which they were obtained.     

Identification of smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta with monoclonal antibody IIG10

We have developed a monoclonal antibody that specifically interacts with a surface antigen of human fibroblasts and smooth muscle cells. The antibody (antibody IIG10) recognizes a polypeptide of molecular mass 330,000, revealed by immunoblotting in fibroblast and smooth muscle cell extract, but not in vascular endothelial cells, peritoneal macrophages, peripheral blood lymphocytes nor hepatocytes. In tissue sections the antibody stained smooth muscle cells of myometrium, aorta and smaller blood vessels, and fibroblasts of connective tissue. Specificity of the antibody was further confirmed by double staining of aorta sections. Antibody IIG10 was used to identify smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta.     

Human smooth muscle VLA-1 integrin: purification, substrate specificity, localization in aorta, and expression during development

A membrane glycoprotein complex was isolated and purified from human smooth muscle by detergent solubilization and affinity chromatography on collagen-Sepharose. The complex was identified as VLA-1 integrin and consisted of two subunits of 195 and 130 kD in SDS-PAGE. Liposomes containing the VLA-1 integrin adhered to surfaces coated with type I, II, III, and IV collagens, Clq subcomponent of the first component of the complement, and laminin. The liposomes specifically adhered to these proteins in a Ca2+, Mg2(+)-dependent manner, but did not bind to gelatin, fibronectin, and thrombospondin substrates. The expression of VLA-1 integrin in different human tissues and cell types, and during aorta smooth muscle development was studied by SDS-PAGE, and subsequent quantitative immunoblotting was performed with antibodies recognizing alpha 1 and beta 1 subunits of the VLA-1 integrin. A high level of VLA- 1 integrin expression was an exceptional feature of smooth muscles. Fibroblasts, endothelial cells, keratinocytes, striated muscles, and platelets contained trace amounts of VLA-1 integrin. In the 10-wk-old human fetal aorta, VLA-1 integrin was found only in smooth muscle cells whereas mesenchymal cells, surrounding aortic smooth muscle cells, were VLA-1 integrin negative. By the 24th wk of gestation, the amount of VLA- 1 integrin was significantly reduced in the aortic media (4.3-fold for alpha 1 subunit and 2.5-fold for beta 1 subunit) compared with that in the 10-wk-old aortic smooth muscle cells. After birth, the expression of VLA-1 integrin increased and in the 1.5-yr-old child aorta the VLA-1 integrin level was almost the same as in adult aortic media. Smooth muscle cells from intimal thickening of adult aorta express five times less alpha 1 subunit of VLA integrin that smooth muscle cells from adult aortic media. In primary culture of aortic smooth muscle cells, the content of the VLA-1 integrin was dramatically reduced and subcultured cells did not contain VLA-1 integrin at all.     

大鼠主动脉内皮细胞和平滑肌细胞的原代培养及生物学特性比较

目的培养大鼠主动脉平滑肌细胞和内皮细胞,细胞纯化与鉴定,比较生物学特性的差异。方法采用血管环贴壁法培养动脉内皮细胞,组织块贴壁法培养动脉平滑肌细胞,并采用有限稀释法挑选内皮细胞单克隆,免疫细胞荧光鉴定二者的特异性标志,相差显微镜观察二者单个细胞及细胞群体在形态上的差异性,CCK-8试剂盒检测细胞的增殖,比较二者对胰酶消化,粘附,冻存后复苏的情况。结果血管环贴壁法成功培养血管内皮细胞,组织块培养法成功培养出血管平滑肌细胞,内皮细胞能够形成单克隆集落,培养的细胞均表达相应的特异性标志,内皮细胞增殖速度和平滑肌细胞有差异,内皮细胞对胰酶的耐受性较差,内皮细胞粘附所需时间短,对冻存后的耐受性较好。结论组织块贴壁法适合内皮细胞和平滑肌细胞的培养,有限稀释法能够纯化原代培养的内皮细胞,大鼠主动脉平滑肌细胞和内皮细胞在细胞形态、增殖、粘附、对胰酶的反应、冻存后复苏均存在差异。     

Characterization of myosin heavy chains in cultured aorta smooth muscle cells. A comparative study

Myosin heavy chains (MHCs) from rat aorta smooth muscle cells were analyzed prior to and after these cells were placed into cell culture using sodium dodecyl sulfate-5% polyacrylamide gels, immunoblots, and two-dimensional peptide maps of tryptic digests. Rat aorta smooth muscle cells prior to culture were found to contain two MHCs (mass = 204 and 200 kDa) which cross-reacted with antibodies raised to smooth muscle myosin, but not with antibodies raised to platelet myosin. Tryptic peptide maps of these two MHCs showed no major differences when compared to each other and to maps of vas deferens and uterus smooth muscle MHCs. When rat aorta smooth muscle cells were placed into culture, the MHCs isolated from the cell extracts differed, depending on whether the cells were rapidly growing or postconfluent. Extracts from log-phase cultures contained predominantly MHCs that migrated more rapidly than smooth muscle myosin in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (mass = 196 kDa) and cross-reacted with antibodies raised to platelet myosin, but not to smooth muscle myosin. Tryptic peptide maps of this MHC were very similar to those obtained with MHCs from non-muscle sources such as platelets and fibroblasts. In contrast, extracts from postconfluent rat aorta cell cultures contained three MHCs (mass = 204, 200, and 196 kDa). Using immunoblots and peptide maps, the fastest migrating MHC was found to be identical to the 196-kDa non-muscle MHC, while the two slower migrating MHCs had the same properties as aorta smooth muscle MHCs prior to culture. These results suggest that smooth muscle cells grown in primary culture contain predominantly (greater than 80%) non-muscle myosin while actively growing, but at a postconfluent stage, contain more equivalent amounts of smooth muscle and non-muscle myosins.     

A membrane potential-sensitive dye for vascular smooth muscle cells assays

Changes in membrane potential of rat aorta smooth muscle cells were investigated using the bis-oxonol sensitive probe DIBAC2(3). We compared the changes in membrane potential induced by a high external KCl concentration in aorta smooth muscle cells from normotensive 2 kidney (2K) and from renal hypertensive 2 kidney-1 clip (2K-1C) rats. The spectral properties of the membrane potential were first characterized in aqueous buffers and in cultured smooth muscle cells from 2K and 2K-1C rat aortas. Fluorescence emission and the images were recorded using a laser scanning confocal microscope. The relationship between fluorescence intensity (FI) and membrane potential (psi(m)) as a function of the increasing extracellular KCl concentration was linear in the 5-40 mmol/L KCl range in both 2K and 2K-1C rat aorta cells. Cell membranes from 2K-1C rat aorta cells were more depolarized (-55 mV) than 2K rat aorta cells (-65 mV). The results show that in 2K-1C aorta cells only 10 mmol/L KCl was needed to induce complete membrane depolarization while in 2K cells 40 mmol/L KCl was needed to induce a similar effect. This study clearly shows that the method is suitable to measure the membrane potential in cultured smooth muscle cells.     

Soluble semicarbazide sensitive amine oxidase (SSAO) catalysis induces apoptosis in vascular smooth muscle cells

Semicarbazide sensitive amine oxidase (SSAO) metabolizes oxidative deamination of primary aromatic and aliphatic amines. It is selectively expressed in vascular cells of blood vessels, but it is also circulating in blood plasma. SSAO activity in plasma is increased in some diseases associated with vascular complications and its catalytic products may cause tissue damage. We examined the effect of the oxidation of the SSAO substrate, methylamine, on cultured smooth muscle cells. Cell incubation with methylamine plus soluble SSAO, contained in bovine serum, resulted toxic to rat aorta A7r5 and human aortic smooth muscle cells, as measured by MTT reduction. This effect was completely reverted by specific SSAO inhibitors, indicating that the toxicity was mediated by the end products generated. Moreover, SSAO-mediated deamination of methylamine induced apoptosis in A7r5 cells, detected by chromatin condensation, Caspase-3 activation, PARP cleavage and cytochrome c release to cytosol. Formaldehyde, rather than H2O2, resulted to be a strong apoptotic inducer to A7r5 cells. Taken together, the results suggest that increased plasma SSAO activity in pathological conditions, could contribute to apoptosis in smooth muscle cells, leading to vascular tissue damage.     

The three-dimensional structure of the tunica intima and tunica media of the human fetal aorta studied by a new method]

A new method is developed for revealing the latent surfaces in the structure of organs by scanning electronic microscopy. The method is based on the treatment of specimens with potassium ethoxide until cells start to appear in the dissociating solution. Using this method, thoracic aorta of nine human fetuses at the stage of 20-28 weeks was studied. Subendothelial intima and media of human fetal aorta contain smooth muscle cells differing by their arrangement, shape and surface microrelief. The intima cells are arranged in a mosaic pattern formed of single cells or cell clusters. By means of cell processes they are connected with each other, as well as with endothelial and smooth muscle cells of the media. Smooth muscle cells in the inner part of the media also have processes and form an open network. Part of the cells penetrate the intima through pores of the inner elastic membrane. In the deeper layers of the media, laterally adjoining spindle-shaped smooth muscle cells are found. It is suggested that the observed cell polymorphism is due mostly to penetration of the media smooth muscle cells into subendothelium and modification of their shape under the effect of the microenvironment.     

Smooth muscle cells in aortic cultures of untreated and cholesterol-fed rabbits

Summary Smooth muscle cells were identified in aortic cultures from which the explants had not been removed. They appeared in cultures from intact aorta within 10 days and in cultures from aorta with plaques in 15 days. Myometrial smooth muscle cells grew within 2–4 days.Differences in growth rate of smooth muscle cells from various sources were explained as were differences in growth of aortic and subcutaneous fibroblasts. Morphologic characteristics of smooth muscle cells and fibroblasts were described in details. Peculiarities of migration of smooth muscle cells in vitro were discussed.This study was supported by grant HE-02534 from the National Heart Institute.     

A comparative study on the metabolic activation of 3,4-benzo[a]pyrene to mutagens by aorta smooth muscle cells of rat and rabbit

The mutagenic activation of benzo[a]pyrene (BaP) after exposure to aorta smooth muscle cells of different origin was examined. Three test systems with different genetic endpoints--sister-chromatid exchange (SCE), gene mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) locus and unscheduled DNA synthesis (UDS)--were used. Treatment of rat and rabbit aorta smooth muscle cells with BaP (1-6 micrograms/ml) resulted in a significant increase of SCEs, HGPRT mutations and UDS. So smooth muscle cells are capable of converting BaP to metabolites with a DNA-damaging action. In order to investigate the relation between the formation of mutagenic BaP metabolites and the susceptibility to atherosclerosis we compared the mutagenic potential of BaP using aorta smooth muscle cells of different species (rat, rabbit) and locations (thoracic and abdominal aorta). Rabbits and abdominal aortas are more susceptible to atherosclerosis than rats and thoracic aortas. The SCE, HGPRT and UDS assays revealed that smooth muscle cells of different origin possessed the same metabolic potential towards BaP. There was no correlation between the mutagenic potency of BaP metabolites and the susceptibility to atherosclerosis. As smooth muscle cells have a low metabolic capacity towards BaP, probably other factors in addition to the metabolic capacity of smooth muscle cells are responsible for species and tissue differences in susceptibility to atherosclerosis.     

The heavy chain of smooth muscle myosin is phosphorylated in aorta cells

The 204-kDa smooth muscle myosin heavy chain (MHC) from rat aorta smooth muscle cells was found to be phosphorylated following isolation of myosin from strips of intact aorta as well as from primary cultures of aorta cells. Two-dimensional maps of the tryptic peptides revealed that the phosphate was confined to only three peptides and gave a similar pattern for the MHC isolated from intact aorta strips and cultured cells. This map was quite different from the phosphopeptide map found for the 196-kDa MHC of nonmuscle myosin isolated from the same cell culture. Smooth muscle MHC purified from primary cell cultures was found to contain approximately 0.7 mol of phosphate/mol of MHC while the nonmuscle MHC contained approximately 0.8 mol of phosphate/mol of MHC. These observations raise the possibility of an additional regulatory mechanism in smooth muscle operating via MHC phosphorylation.     

Presence of epidermal growth factor receptors and influence of epidermal growth factor on proliferation and aging in cultured smooth muscle cells.

Epidermal growth factor (EGF) at nanomolar concentrations stimulated DNA synthesis in confluent, serum-starved cultures of calf aorta and human uterine smooth muscle cells. Stimulation of DNA synthesis in lens epithelial cells was studied for comparison. L and D-ascorbic acid potentiated the effect of serum and EGF on DNA synthesis in calf aorta cells. In contrast L-ascorbic acid had minimal potentiating effect with serum and no effect with EGF present along with serum on DNA synthesis in human uterine smooth muscle and rabbit lens epithelial cells. EGF and ascorbic acid increased cell number when added to stationary phase cultures. Specific binding of 125I-labelled EGF to smooth muscle cells was demonstrated. Receptor concentration in calf-aorta smooth muscle cells was higher in dense cultures compared to sparse cultures. The time course of binding and dissociation of 125I-labelled EGF was similar in "dense" and "sparse" cultures. Human uterine smooth muscle cells in culture exhibited a finite lifespan. There was no stimulation of DNA synthesis in response to serum and EGF in cells of high population doubling level (PDL); although 125I-labeled EGF binding was higher in old cells (high PDL) compared to young cells (low PDL). This increase in binding was shown to be due to changes in the concentration of receptors without changes in their affinity for EGF.