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.

p63/MT1-MMP axis is required for in situ to invasive transition in basal-like breast cancer.

The transition of ductal carcinoma in situ (DCIS) to invasive breast carcinoma requires tumor cells to cross the basement membrane (BM). However, mechanisms underlying BM transmigration are poorly understood. Here, we report that expression of membrane-type 1 (MT1)-matrix metalloproteinase (MMP), a key component of the BM invasion program, increases during breast cancer progression at the in situ to invasive breast carcinoma transition. In the intraductal xenograft model, MT1-MMP is required for BM transmigration of MCF10DCIS.com breast adenocarcinoma cells and is overexpressed in cell clusters overlying focal BM disruptions and at the invasive tumor front. Mirrored upregulation of p63 and MT1-MMP is observed at the edge of MCF10DCIS.com xenograft tumors and p63 is required for induction of MT1-MMP-dependent invasive program in response to microenvironmental signals. Immunohistochemistry and analysis of public database reveal that p63 and MT1-MMP are upregulated in human basal-like breast tumors suggesting that p63/MT1-MMP axis contributes to progression of basal-like breast cancers with elevated p63 and MT1-MMP levels.

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.

Challenges in the stratification of breast tumors for tailored therapies.

Studying the molecular stratification of breast carcinoma is a real challenge considering the extreme heterogeneity of these tumors. Many patients are now treated following recommendation established at several NIH and St Gallen consensus conferences. However a significant fraction of these breast cancer patients do not need adjuvant chemotherapies while other patients receive inefficacious therapies. High density gene expression arrays have been designed to attempt to establish expression profiles that could be used as prognostic indicators or as predictive markers for response to treatment. This review is intended to discuss the potential value of these new indicators, but also the current weaknesses of these new genomic and bioinformatic approaches. The combined analysis of transcriptomic and genomic alteration data from relatively large numbers of well annotated tumor specimens may offer an opportunity to overcome the current difficulties in validating recently published non overlapping gene lists as prognostic or therapeutic indicators. There is also hope for identifying and deciphering signal transduction pathways driving tumor progression with newly developed algorithms and semi quantitative parameters obtained in simplified in vitro or in vivo models for specific transduction pathways.

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.

Myoepithelial cell differentiation in the developing mammary gland: progressive acquisition of smooth muscle phenotype.

The most important portion of the mammary gland development occurs postnatally, with distinct periods of intensive morphogenesis taking place between birth and puberty and during pregnancy and lactation. To characterize the differentiation process of mammary myoepithelial cells, we have studied the expression patterns of several smooth muscle phenotypic markers, including contractile proteins, alpha-smooth muscle-actin (alpha-SM-actin), smooth muscle myosin heavy chains (SM-MHC), and calponin; components of cell-extracellular matrix adherens junctions, phosphoglucomutase-related protein (PGM), vinculin variants, integrin subunits; and laminin variant chains in the developing rat mammary gland using immunofluorescence microscopy. alpha-SM-actin- and SM-MHC-positive cells were found first in newborn animals, while calponin, PGM and alpha1 integrin subunit began to be expressed in prepubertal animals (1.5 weeks). Vinculin, beta1 and alpha3 integrin subunits were largely confined to the basal cell layer at all developmental stages examined with greater staining starting at 1.5 weeks. Meta-vinculin was identified only in myoepithelial cells of the lactating gland. gamma1 laminin chain was present in the mammary gland basement membrane throughout development, while the beta2 chain was revealed in 3-week-old animals and accumulated later in postpubertal animals (7 weeks). Similarly, beta2 laminin chain was absent from the forming alveoli basement membrane in pregnant rats and started to accumulate in the lactating gland. In addition to temporal changes, we have observed spatial differences in the distribution of the phenotypic markers. Both in pre- and in postpubertal animals, alpha-SM-actin- and SM-MHC-positive cells of the growing ductal ends contained low amounts if any of calponin, PGM, and beta2 laminin chain. We conclude that during postnatal development, mammary myoepithelial cells progressively acquire a differentiated phenotype as revealed by the expression of various smooth muscle markers. Maturation of the myoepithelial cells is accompanied by upregulation of the smooth muscle integrin expression followed by accumulation of beta2-containing laminin variant. Thus, changes in adhesion system parallel with the myoepithelial cell differentiation.

Tumor progression: the role of cadherins and integrins.

Established cadherin and integrin-containing adherens junctions maintain the integrity of normal tissues. Signalling via adhesion-molecule systems is an important factor in the control of cellular growth and differentiation. In transformed cells, destructive changes in the adhesion systems lead to abnormal relationships among neighbouring cells and the extracellular matrix. Adhesion molecules may prevent tumour progression by firmly attaching cells to each other, and by anchoring them in the extracellular matrix. In addition, cadherins and integrins may have a direct role in tumour suppression by participating in growth control. Dissemination of cancer cells, i.e. invasion and metastasis, requires movement of cells, as well as adhesion to extracellular matrices and to other cells. Particular integrins have been implicated in several aspects of this multistep process. In this article, the data on the possible roles of cadherins and integrins in tumor progression are summarized.

Adhesion systems in normal breast and in invasive breast carcinoma.

To analyze the role of various elements of the adhesion system in the organization of the normal mammary gland and in breast carcinoma, we have studied simultaneously the expression of integrins, E- and P-cadherins, and cytoplasmic constituents of adherens junctions. In the normal gland, E-cadherin and alpha-catenin are present in luminal epithelial and myoepithelial cells, whereas integrins are more abundant in acinar epithelial and in myoepithelial cells. We demonstrate here that, in addition, myoepithelial cells express much more vinculin and alpha-actinin than luminal epithelial cells, whereas talin and focal adhesion kinase (pp125FAK) are restricted to the basal cell layer. In invasive carcinoma, E-cadherin is usually present although often in reduced amount; different integrin subunits are expressed either by a fraction or by all of the cells or are absent. However, the cytoplasmic components of adherens junctions, such as alpha-catenin, vinculin, alpha-actinin, talin, and pp125FAK, are expressed at low levels or cannot be detected in the carcinoma cells. Our data suggest that 1), in the normal mammary gland, the myoepithelial cells, being particularly rich in integrins and cytoplasmic components of the adherens junctions, play an important role in the maintenance of tissue integrity; 2), in invasive carcinoma, cell aggregates may be maintained due to varying levels of expression of E-cadherin and/or integrins; and 3), interaction of the transmembrane adhesion molecules with the cytoskeleton in carcinoma may be impaired as revealed by reduced levels of expression of alpha-catenin, vinculin, alpha-actinin, talin, and pp125FAK. Importantly, carcinoma cells, when exposed to stroma during invasion, do not acquire the adhesion apparatus characteristic of normal cells in contact with the extracellular matrix.

Arterial smooth muscle cell phenotype in stroke-prone spontaneously hypertensive rats.

The aim of this study was to determine the phenotype of smooth muscle cells in the arteries of chronically hypertensive animals and to analyze the effects of treatments known to increase the survival of the animal without a clear effect on its hypertensive state. Stroke-prone spontaneously hypertensive rats (SHRSP) kept on a 1% sodium drinking solution were untreated or treated with one of two diuretics, indapamide (3 mg/kg per day) or hydrochlorothiazide (20 mg/kg per day), from 6 to 13 weeks of age. Phenotype was characterized by the immunolabeling of arteries with antibodies raised against a cellular form (EIIIA) of fibronectin, alpha-smooth muscle actin, and nonmuscle myosin. We demonstrated that phenotypes of smooth muscle cells of the SHRSP differ from those found in Wistar-Kyoto rats. The difference in phenotype is specific for the vessel type: ie, an increased expression of nonmuscle myosin in the aorta and of both EIIIA fibronectin and nonmuscle myosin in the coronary arteries. The two diuretics (1) had no effect on blood pressure, (2) prevented or did not prevent the increase in medial thickness, and (3) prevented changes in both smooth muscle cell phenotype and ischemic tissular lesions. Taken together, the results suggest that in SHRSP the changes in the phenotype of smooth muscle cells and the thickness of arteries are unrelated events. We propose that the maintenance of the contractile phenotype of the arterial smooth muscle cells could be an essential parameter involved in the prevention of the deleterious consequences characteristic of a severe hypertensive state.

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.

Laminin variants and integrin laminin receptors in developing and adult human smooth muscle.

Distribution of laminin variant chains and of laminin-binding beta 1-associated alpha integrin subunits was studied in human arterial and visceral smooth muscle during development using the immunofluorescence method. In aortic media of 10-week-old fetuses (early fetal stage of development), B1, B2, and A chains were found, and at 27 weeks of gestation (late fetal step), in addition, the S chain was revealed. In the major part of adult aortic media, staining for the B1 chain was substituted by staining for the S chain. A reasonable explanation for this is that the basement membrane of arterial SMCs contains the A-B1-B2 laminin trimer at the early fetal stage of development, a mixture of A-B1-B2 and A-S-B2 variants during the late fetal period, and the A-S-B2 form in adult media. In the intimal thickening of adult arteries, staining for A and S chains was decreased, and the B1 chain was detected, thus suggesting coexistence of A-B1-B2 and A-S-B2 variants similar to the fetal aorta. These data led us to propose that maturation of arterial SMCs is accompanied by a switch from expression of A-B1-B2 to A-S-B2 laminin, the only protein variant associated with the differentiated SMC phenotype. The M chain was not detected in fetal and adult arterial smooth muscle. In differentiated visceral SMCs from adult colon, in contrast to arterial medial SMCs, B1, S, B2, and A chains, i.e., both B1 chain- and S chain-containing laminin molecules, were present. Similar to vascular smooth muscle, the expression of A and S laminin chains in SMCs of the intestine was developmentally regulated. Of the four beta 1-associated alpha integrin subunits that have been shown to recognize laminin (alpha 1, alpha 2, alpha 3, and alpha 6), only alpha 1 was revealed during the early fetal stage of development in smooth muscle of the arteries and digestive tract. alpha 3 appeared in aortic media only during the late fetal period. In adult arterial media and smooth muscle of the colon, alpha 1 and alpha 3 were the major potential laminin-binding integrins. The spatial-temporal distribution of laminin variants in developing and adult smooth muscle suggests that a variant laminin (possibly A-S-B2) interacting with its receptor (alpha 1 beta 1 or alpha 3 beta 1) may be important for maintenance of the differentiated SMC phenotype in vivo.

Diuretic effects on cardiac hypertrophy in the stroke prone spontaneously hypertensive rat.

OBJECTIVE: The aim was to compare the effects of two diuretics, indapamide and hydrochlorothiazide, on cardiac hypertrophy in stroke prone spontaneously hypertensive rats (SHR-SP). METHODS: Six week old SHR-SP, on a 1% sodium chloride water intake, were treated with oral indapamide (3 mg.kg-1 x d-1) or hydrochlorothiazide (20 mg.kg-1 x d-1) over a 44 d period. The hypertrophic process was evaluated by classical indices and by the morphological analysis of myocyte cross sectional area, coronary artery thickness, and immunohistochemical analysis of interstitial fibrosis. RESULTS: In the untreated SHR-SP on 1% sodium chloride, all animals developed severe hypertension and cardiac hypertrophy when compared to normotensive salt loaded WKY by 13 weeks of age. In salt loaded SHR-SP treated with indapamide or hydrochlorothiazide, systolic blood pressure was moderately decreased by the end of the treatment when compared with untreated SHR-SP, at 259(7) and 245(7) mm Hg respectively, v 300(11) mm Hg, p < or = 0.05. Myocyte enlargement appears to be the main feature involved in the development of cardiac hypertrophy in the SHR-SP. By the end of treatment both indapamide and hydrochlorothiazide prevented the development of cardiac hypertrophy evaluated by heart weight to body weight ratio [4.69(0.07) and 4.61(0.08) respectively, v 5.39(0.13), p < or = 0.001] and myocyte hypertrophy (-33% and -21% of the SHR-SP values, p < or = 0.001). Myocardial interstitial fibrosis and perivascular fibrosis were practically absent in the two treated groups. CONCLUSIONS: Our results allow the characterisation of SHR-SP cardiac hypertrophy and indicate that the two types of chronic diuretic treatment prevent SHR-SP cardiac hypertrophy with a drug specific efficiency.