p53 deficiency induces cancer stem cell pool expansion in a mouse model of triple-negative breast tumors.

Triple-negative breast cancer is a heterogeneous disease characterized by the expression of basal cell markers, no estrogen or progesterone receptor expression and a lack of HER2 overexpression. Triple-negative tumors often display activated Wnt/ß-catenin signaling and most have impaired p53 function. We studied the interplay between p53 loss and Wnt/ß-catenin signaling in stem cell function and tumorigenesis, by deleting p53 from the mammary epithelium of K5ΔNßcat mice displaying a constitutive activation of Wnt/ß-catenin signaling in basal cells. K5ΔNßcat transgenic mice present amplification of the basal stem cell pool and develop triple-negative mammary carcinomas. The loss of p53 in K5ΔNßcat mice led to an early expansion of mammary stem/progenitor cells and accelerated the formation of triple-negative tumors. In particular, p53-deficient tumors expressed high levels of integrins and extracellular matrix components and were enriched in cancer stem cells. They also overexpressed the tyrosine kinase receptor Met, a feature characteristic of human triple-negative breast tumors. The inhibition of Met kinase activity impaired tumorsphere formation, demonstrating the requirement of Met signaling for cancer stem cell growth in this model. Human basal-like breast cancers with predicted mutated p53 status had higher levels of MET expression than tumors with wild-type p53. These results connect p53 loss and ß-catenin activation to stem cell regulation and tumorigenesis in triple-negative cancer and highlight the role of Met signaling in maintaining cancer stem cell properties, revealing new cues for targeted therapies.

Signaling events mediated by α3ß1 integrin are essential for mammary tumorigenesis.

The constitutive activation of ß-catenin signaling in the mammary basal epithelial cell layer in transgenic K5ΔNßcat mice leads to basal-type tumor development. Integrins of the ß1 family and integrin-mediated signaling events have an important role in breast tumor growth and progression. We show here that the deletion of α3ß1 integrin, a major laminin receptor, from the basal layer of the mammary epithelium of K5ΔNßcat mice completely prevented the tumorigenesis induced by ß-catenin signaling. Moreover, the depletion of α3ß1 integrin from a spontaneously transformed mouse mammary basal epithelial cell line (MEC) prevented the cells from forming colonies in soft agar and greatly reduced tumor development in orthotopic grafts. Inhibition of the integrin signaling intermediates Rac1 or PAK1 (P21-activated Kinase 1) in MEC affected tumor cell growth in soft agar, whereas the expression of activated forms of these effectors in α3-depleted cells rescued the capacity of these cells to grow in non-adherent conditions. Similarly, the tumorigenic potential of α3-depleted cells was restored by the expression of activated PAK1, as assessed by orthotopic transplantation assay. In three-dimensional Matrigel culture, MEC survival and proliferation were affected by the depletion of α3ß1 integrin, which also significantly decreased the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK). Our data suggest that the activation of signaling cascades downstream from α3ß1 and involving the Rac1/PAK1 pathway, MAPK and JNK, promotes prosurvival and proproliferative signals required for the malignant growth of basal mammary epithelial cells, providing further insight into the molecular mechanisms underlying breast cancer initiation and progression.

beta-catenin-dependent and -independent effects of DeltaN-plakoglobin on epidermal growth and differentiation.

Both beta-catenin and plakoglobin can stimulate the expression of Lef/Tcf target genes in vitro. beta-Catenin is known to associate with Lef/Tcf factors and to participate directly in transactivation in vivo, whereas the role of plakoglobin in transcriptional regulation has been less studied. To analyze the functions of plakoglobin in vivo, we generated transgenic mice expressing in the epidermis N-terminally truncated plakoglobin (DeltaN122-PG) lacking the glycogen synthase kinase 3beta phosphorylation sites and therefore protected against degradation (transgenic line K5-DeltaN122-PG). The expression of DeltaN122-PG led to the formation of additional hair germs, hyperplastic hair follicles, and noninvasive hair follicle tumors, a phenotype reminiscent of that induced by expression of N-terminally truncated beta-catenin. However, if expressed in beta-catenin-null epidermis, DeltaN122-PG did not induce new hair follicle germs and follicular tumors. Thus, DeltaN122-PG cannot substitute for beta-catenin in its signaling functions in vivo and the phenotype observed in K5-DeltaN122-PG mouse skin must be due to the aberrant activation of beta-catenin signaling. On the other hand, the expression of DeltaN122-PG in beta-catenin-null skin significantly increased the survival rate of mutant mice, rescued differentiation, and limited excessive proliferation in the interfollicular epidermis, suggesting that plakoglobin may be involved in the intracellular signaling events essential for epidermal differentiation.

Growth defects induced by perturbation of beta1-integrin function in the mammary gland epithelium result from a lack of MAPK activation via the Shc and Akt pathways.

Adhesion to extracellular matrix (ECM) induces intracellular signals that modulate cell proliferation, survival and differentiation. To study signalling events triggered by cell-ECM interactions in vivo we used transgenic mice exhibiting reduced mammary epithelial cell proliferation and increased apoptosis rates during the growth phase in pregnancy and lactation due to expression of a beta1-integrin dominant-negative mutant in the mammary gland epithelium. Here we show that ERK and JNK MAPKs were markedly less activated in lactating transgenic glands thereby accounting for the growth defects. The FAK pathway was not affected suggesting a mechanism of activation additional to the ECM signal. On the contrary, the significant decrease of Shc phosphorylation, Grb2 recruitment and the reduced phosphorylation level of Akt Thr308 and Akt substrates FKHR and Bad detected in transgenic glands show that activation of the Shc and the Akt pathways require intact cell-ECM interactions. These results provide an insight into the mechanisms of growth control by integrin-mediated adhesion that operate in vivo.

Development of mammary gland requires normal beta 1-integrin function.

To study the role of beta 1-integrins in mammary gland development we have generated transgenic mice expressing a dominant negative mutant of the beta 1-integrin chain in the mammary epithelium. The transgenic glands presented a delayed development in pregnancy and lactation due to decreased epithelial cell proliferation and increased apoptosis, whereas at the beginning of lactation, expression of milk proteins, WAP and beta-casein was diminished. In correlation with transgene expression, the basement membrane component, laminin, and the beta 4 integrin were accumulated at the lateral surface of luminal epithelial cells, revealing defects in polarization. Our data show that beta 1-integrins are involved in vivo in the control of proliferation, apoptosis, differentiation, and maintenance of baso-apical polarity of mammary epithelium.

Cell-extracellular matrix interactions and EGF are important regulators of the basal mammary epithelial cell phenotype.

The mammary epithelium is composed of a luminal epithelium and a basal layer containing myoepithelial cells and undifferentiated precursors. Basal cells express specific protein markers, such as keratin 14 (K14) and P-cadherin. To study the factors that regulate the basal mammary epithelial cell phenotype, we have established two clonal derivatives of the mouse HC11 cell line, BC20 and BC44, expressing high levels of K14 and P-cadherin. Unlike the parental HC11 cells, these basal cells did not produce beta-casein in response to lactogenic hormone treatment; however their phenotype appeared to be plastic. Cultured in EGF-free medium, they exhibited enhanced cell-extracellular matrix adhesions and deficient cell-cell junctions, whereas long-term treatment with EGF induced a decrease of focal contact number and establishment of cell-cell junctions, resulting in downregulation of K14 and P-cadherin expression at the protein and mRNA levels. To determine whether cell-extracellular matrix interactions mediated by integrins have a role in the regulation of the expression of K14 and P-cadherin, the amounts of transcripts for the two proteins were analysed in the basal cells, which were plated on the function-blocking antibodies against beta1 and alpha6 integrin chains, on fibronectin and on laminin 5. The amount of P-cadherin transcript was 2- to 4-fold higher in cells plated on the function-blocking anti-integrin antibodies and on the extracellular matrix proteins, as compared to cells plated on poly-L-lysine, whereas the K14 transcript levels were not significantly modified in response to adhesion. The data demonstrate that integrin-mediated cell interaction with extracellular matrix is directly implicated in the control of P-cadherin expression, and that EGF and cell-extracellular matrix adhesion events are important regulators of the basal mammary epithelial cell phenotype.

Perturbation of beta1-integrin function alters the development of murine mammary gland.

The expression of a transgene coding for a chimeric molecule, containing the cytoplasmic and transmembrane domains of the beta1-integrin chain and the extracellular domain of the T-cell differentiation antigen CD4, was targeted to the mouse mammary gland by the mouse mammary tumor virus (MMTV) promoter. The chimera does not interact with the extracellular ligands; however, its expression in cultured cells was shown to interfere with focal adhesion kinase (FAK) phosphorylation following ligation of endogenous beta1-integrin. Therefore, expression of the transgenic protein on the cell surface should uncouple adhesion from intracellular events associated with the beta1-cytoplasmic domain and thus perturb beta1-integrin functions. Although most of the transgenic females were able to lactate, their mammary glands had a phenotype clearly distinct from that of wild-type mice. At mid-pregnancy and the beginning of lactation, transgenic glands were underdeveloped and the epithelial cell proliferation rates were decreased, while the apoptosis levels were higher than in wild-type glands. In lactation, the amounts of the whey acidic protein (WAP) and beta-casein gene transcripts were diminished, and the basement membrane component, laminin and the beta4-integrin chain accumulated at the lateral surface of luminal epithelial cells, revealing defects in polarization. Our observations prove that in vivo, beta1-integrins are involved in control of proliferation, apoptosis, differentiation and maintenance of baso-apical polarity of mammary epithelial cells, and therefore are essential for normal mammary gland development and function.

Cytokeratin expression in human arteries pertinent to intimal thickening formation in the ductus arteriosus.

Expression of epithelial cytokeratins type 8, 18 and 19 can be used to study smooth muscle cell differentiation during development. We studied the differentiation of smooth muscle cells in the ductus arteriosus before and during intimal thickening and compared the changes occurring in this vessel with the adjoining elastic ascending and descending aorta and the pulmonary trunk. The ductus arteriosus, a vessel connecting the pulmonary trunk and the aorta during fetal life, constricts shorty after birth and eventually closes. Effective closure occurs only in the case of well developed intimal thickening. Cytokeratin expression during fetal development was greatest in the media of the ascending aorta and pulmonary artery, while in the ductus and descending aorta cytokeratin staining was slight. These results suggest that ductus smooth muscle cells and the smooth muscle cells of the descending aorta show a more advanced differentiation as compared to the ascending aorta and pulmonary artery. At neonatal stages cytokeratin expression in the descending aorta, pulmonary artery and the ascending aorta had disappeared as was expected with increased differentiation. In the neonatal ductus arteriosus reexpression of cytokeratins was found in cell clusters in the hyaluronic acid rich environment of the intimal thickening and in the inner media. Reexpression of cytokeratins, especially when organized in clusters, may reflect changes in gene regulation. Therefore the clusters of cytokeratin positive cells in the ductus may be indicative of extensive changes, occurring during closure of this vessel in the neonatal period, in which inner media and intima are especially involved.

Differentiation, dedifferentiation, and apoptosis of smooth muscle cells during the development of the human ductus arteriosus.

Differentiation of vascular smooth muscle cells (SMCs) is characterized by several molecular transitions. As differentiation proceeds, proteins of the cytoskeletal and contractile apparatus, such as alpha-smooth muscle actin, smooth muscle myosin, calponin, and heavy caldesmon, and the expression of the membrane-related protein smooth muscle phosphoglucomutase-related protein increase, whereas the expression of other proteins, such as fibronectin splice variants with extradomains A (EDA) and B (EDB), decreases. In this study, we investigated the differentiation of the SMCs of the ductus arteriosus during the development of intimal thickening. Ascending and descending aortas of the same age were used for comparison because these vessels lack intimal thickening. In the fetal ductus arteriosus, a relatively early differentiation of the contractile apparatus was observed compared with the ascending and descending aortas. EDA and EDB expression was already low, being similar in the ductus and descending aorta and even lower in the ascending aorta. In the neonatal ductus, SMCs of the media and outer intima were well differentiated and comparable with SMCs of the ascending aorta. Dedifferentiated SMCs, with a low expression of cytoskeletal and contractile proteins and a high expression of EDA and EDB, were found in regions in the inner intima that show features of progression of intimal thickening and in areas of cytolytic necrosis in the media. With a technique using in situ end labeling of DNA fragments, we found extensive apoptosis in the area of cytolytic necrosis and to a lesser extent in these areas of the inner intima. In conclusion, SMCs of the fetal ductus arteriosus have an advanced differentiation of the contractile apparatus compared with the adjacent aorta. Reexpression of fetal characteristics is seen in a number of cells in inner intima and media of the neonatal ductus arteriosus. The finding of apoptosis in these areas suggests that dedifferentiation and apoptosis are associated processes that may play a role in vascular remodeling.

Myosin heavy-chain isoform composition and distribution in developing and adult human aortic smooth muscle.

The myosin heavy-chain (MHC) composition of developing and adult human aortic smooth muscle (SM) was studied by SDS-polyacrylamide gel electrophoresis, Western blotting and indirect immunofluorescence using a panel of anti-MHC antibodies. On 5% SDS gels, three bands of 204, 200 and 196 kDa apparent molecular mass were identified in fetal, infant and adult stages of development. In the extracts from thoracic aorta (upper level), the 204, and 200-kDa bands (designated as SM-1 and SM-2, respectively) were recognized by SM-G4 and SMMS-1 antibodies, raised against a SM antigen, whereas the 196-kDa band was reactive with nonmuscle (NM)-F6 and NM-G2 antiplatelet MHC antibodies. Western blotting and immunofluorescence tests performed on bovine brain and other human NM tissues using NM-F6 and NM-G2 indicated that antigenic targets of the two antibodies resembled that of so-called IIB and IIA NM myosin found in the bovine system, respectively. In the aortic media, SM-1 was expressed throughout development, while SM-2 was upregulated during late fetal and postnatal development. Similarly, the 196-kDa band showed two distinct patterns of immunoreactivity with the anti-NM-MHC antibodies: with NM-G2, antigenicity was equal at all the developmental stages examined, whereas with NM-F6, it diminished during postnatal development. In the upper level, the cellular distribution of NM-G2 and NM-F6 immunoreactivities was similar in the early fetus but quite distinct at later stages of development. In infant and adult subjects, SM cells (SMC) reactive with NM-F6 accumulated predominantly within the intimal layer as well as in some areas of the underlying media as cell foci, whereas NM-G2 homogeneously stained the two layers. In the aorta near the diaphragm (lower level), both antibodies stained the thickened intima but not the underlying media. These data are consistent with the existence of developmental, stage-specific molecular and cellular transitions during vascular SMC maturation in human aortic media. In addition, these data suggest that IIB-like myosin may be expressed in SMC involved specifically in intimal thickening.

Fibronectin and integrins in development.

Extracellular matrix glycoprotein fibronectin and integrins, transmembrane receptors for extracellular matrix proteins, have been implicated in morphogenesis, cytodifferentiation and organogenesis. During development, embryonic cells move on or through fibronectin-rich matrices, which support migration and contain the guidance cues that direct the cells along their migratory pathways. Integrins are involved in different aspects of the movement, including recognition of the pathway, transient attachment to the substrate and cytoskeleton rearrangement in the moving cells as well as organization of the matrix during migratory pathway formation. Differentiated tissues exhibit a characteristic and often rather limited set of integrins. On the contrary, during development, tissue repertoire of integrins changes, and integrins that are absent from the adult differentiated tissue may appear transiently and be expressed only during certain developmental stages. These periods of transient expression coincide with cell movement, differentiation and maturation of cells and can be correlated with changes of environment, i.e. neighboring cells, composition and structure of the extracellular matrix. In this article we discuss the recent data on the roles of fibronectin-integrin interactions in directed cell migration in embryos and on the distribution and possible functions of integrins in developing tissues.

Expression of smooth muscle-specific proteins in myoepithelium and stromal myofibroblasts of normal and malignant human breast tissue.

The expression of several differentiation markers in normal human mammary gland myoepithelium and in certain stromal fibroblasts ("myofibroblasts") associated with breast carcinomas was studied by immunofluorescence microscopy of frozen sections. Several antibodies to smooth muscle-specific proteins (smooth muscle alpha-actin, smooth muscle myosin heavy chains, calponin, alpha 1-integrin, and high molecular weight caldesmon) and to epithelial-specific proteins (cytokeratins, E-cadherin, and desmoplakin) were used to show that myoepithelial cells concomitantly express epithelial and smooth muscle markers whereas adjacent luminal cells express only epithelial markers. The same antibodies were used to establish that stromal myofibroblasts exhibit smooth muscle phenotypic properties characterized by the expression of all the smooth muscle markers examined except for high molecular weight caldesmon. In addition, both myoepithelium and myofibroblasts show a significant degree of heterogeneity in smooth muscle protein expression. Thus, myoepithelial cells and stromal myofibroblasts are epithelial and mesenchymal cells, respectively, which coordinately express a set of smooth muscle markers while maintaining their specific original features. The dual nature of myoepithelial cells and the phenotypic transition of fibroblasts to myofibroblasts are examples of the plasticity of the differentiated cell phenotype.

Phenotypic changes of human smooth muscle cells during development: late expression of heavy caldesmon and calponin.

Expression of the regulatory contractile proteins, heavy caldesmon (h-caldesmon) and calponin was studied in human aortic smooth muscle cells (SMCs) during development and compared with the expression of alpha-SM-actin and smooth muscle-myosin heavy chain (SM-MHCs). For this study, novel monoclonal antibodies specific to SM-MHCs, h-caldesmon, and calponin were developed and characterized. Aortic SMCs from fetuses of 8-10 and 20-22 weeks of gestation express alpha-SM-actin and SM-MHCs, but neither h-caldesmon nor calponin were expressed as demonstrated by immunoblotting and immunofluorescence techniques. In the adult aortic tunica media, SMCs contain all four markers. Thus, the expression of calponin, similar to the expression of alpha-SM-actin, SM-MHCs, and h-caldesmon, is developmentally regulated in aortic SMCs. In the adult aortic subendothelial (preluminal) part of tunica intima, numerous cells containing SM-MHCs, but lacking h-caldesmon and calponin, were found. These results illustrate the similarity of SMCs from intimal thickenings and immature (fetal) SMCs. Expression of contractile proteins in the developing SMCs is coordinately regulated; however, distinct groups of proteins appear to exist whose expression is regulated differently. Actin and myosin, being major contractile proteins, also play a structural role and appear rather early in development, whereas caldesmon and calponin, being involved in regulation of contraction, can serve as markers of higher SMC differentiation steps. In contrast, h-caldesmon and calponin were already present in visceral SMCs (trachea, esophagus) of the 10-week-old fetus. These results demonstrate that the time course of maturation of visceral SMCs is different from that of vascular SMCs.

Developmental changes in expression of adhesion-mediating proteins in human aortic smooth muscle.

Phenotypic variability of vascular smooth muscle cells (SMCs) can serve as a good model for studying the mechanisms regulating the expression of adhesion-mediating proteins. To describe phenotypic changes of human aortic SMCs, we have studied the expression of cytodifferentiation-related adhesion-mediating proteins in samples of media from fetal, child and adult human aorta, and in subendothelial intima of normal and atherosclerotic aorta. We have shown that during prenatal and post-natal development vascular SMCs co-ordinately change several times the expression of certain differentiation-related proteins. Our data show the existence of certain groups of proteins whose expression during smooth muscle development might be controlled by two basic mechanisms: selection of genes to be expressed at particular developmental stages and generation of several different protein variants from a single gene via alternative RNA splicing.

Phenotypic changes of human aortic smooth muscle cells during development and in the adult vessel.

To characterize phenotypic expression of human aortic smooth muscle cells (SMCs), we have studied the content of cytodifferentiation-related cytoskeletal proteins, and of fibronectin (FN) variants in the samples of media from the fetal, child, and adult aorta and in the subendothelial intima of the normal and atherosclerotic aorta. Mature SMCs from the adult aortic media contained high amounts of alpha-SM-actin, SM-myosin heavy chains, meta-vinculin, and 150 kDa caldesmon. Cytokeratin 8 and extra domain-containing variants of FN (A-FN and B-FN) were not found in these cells. The SMCs from the aortic media of 10-wk-old fetus contained low amounts of the SM markers, expressed cytokeratin 8, A-FN, and B-FN. In 25-wk-old fetus, as well as in 2- and 6-mo-old child, aortic medial SMCs expressed an intermediate phenotype, and only in 18-mo-old child were the cells found to be similar to those from adult media. SMCs from the normal adult subendothelial intima contained reduced amounts of meta-vinculin and of 150 kDa caldesmon, and they expressed A-FN. In addition, the SMCs from atherosclerotic fibrous plaque contained a decreased proportion of alpha-SM-actin and of SM-myosin heavy chains, whereas cytokeratin 8 was found. Therefore we conclude that the SMCs from intimal thickenings appear to express a less mature phenotype than that of the medial cells from adult aorta. Rather, these SMCs contain reduced amounts of the SM markers and express proteins typical of the fetal SMC phenotype, A-FN and cytokeratin 8.

Expression of cytokeratin 8 in human aortic smooth muscle cells.

An immunofluorescence method was used to study the expression of cytokeratin 8 in human aortic smooth muscle cells (SMCs) during prenatal development and in atherosclerotic plaques. Aortic SMCs from a 10-wk-old fetus contained cytokeratin 8 in additional to vimentin and a small amount of desmin, whereas, in the cells from a 25-wk-old fetus, cytokeratin 8 was not detected. Cytokeratin 8 was found in the SMCs from intimal thickenings, fatty streaks, and atherosclerotic fibrous plaques. Clusters of cytokeratin 8-positive cells were more abundant in rather advanced lesions (fibrous plaques) that contained at least some amount of lipid. Expression of cytokeratin 8 in the cells of human atherosclerotic lesions probably reflects general rearrangement of gene expression in the intimal cells.

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.

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.

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.