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Phenotypic reversion of smooth muscle cells in hybrid vascular prostheses.

Our purpose was to evaluate whether or not and when phenotypic modulation of smooth muscle cells (SMCs) in hybrid vascular prostheses preincorporated with SMCs occurs upon implantation. Two types of hybrid vascular grafts incorporated with vascular cells derived from canine jugular veins were prepared: grafts containing a collagen gel layer covered with an endothelial monolayer at the luminal surface (Model I graft) and those containing an endothelial monolayer and SMC multilayer (Model II graft). They were bilaterally implanted into carotid arteries of the same dogs from which the cells had been harvested for 2 wk (n = 3) and 12 wk (n = 3). The time-dependent changes in populations of three SMC phenotypes (synthetic, intermediate, and contractile) in the neoarterial layers were quantified by morphometric evaluation using a transmission electron microscope in hybrid vascular grafts. Before implantation, all the SMCs were of the synthetic phenotype. In Model II grafts at 2 wk, synthetic and intermediate SMCs were dominant especially in the luminal layer. On the other hand, neoarterial layers at 12 wk were dominated by contractile SMCs, which were evenly distributed throughout the entire neoarterial tissues. A markedly delayed phenotypic reversion was noted for the Model I grafts at 12 wk. In the hybrid grafts, during about 3 mo of implantation, neoarterial SMCs transformed from the synthetic to the contractile phenotypes, which was promoted by SMC incorporation.

Animals↗

Expression of adhesion molecule T-cadherin is increased during neointima formation in experimental restenosis.

Phenotypic modulation, migration and proliferation of vascular smooth muscle cells (SMCs) are major events in restenosis after percutaneous transluminal angioplasty. Surface cell adhesion molecules, essential to morphogenesis and maintenance of adult tissue architecture, are likely to be involved, but little is known about cell adhesion molecules expressed on SMCs. T-cadherin is a glycosyl phosphatidylinositol-anchored member of the cadherin superfamily of adhesion molecules. Although highly expressed in vascular and cardiac tissues, its function in these tissues is unknown. We previously reported increased expression of T-cadherin in intimal SMCs in atherosclerotic lesions and proposed a role for T-cadherin in phenotype control. Here we performed immunohistochemical analysis of spatial and temporal changes in vascular T-cadherin expression following balloon catheterisation of the rat carotid artery. T-cadherin expression in SMCs markedly increases in the media early (1-4 days) after injury, and later (day 7-28) in forming neointima, especially in its preluminal area. Staining for monocyte/macrophage antigen ED-1, proliferating cell nuclear antigen and smooth muscle alpha-actin revealed that spatial and temporal changes in T-cadherin level coincided with the peak in cell migration and proliferation activity during neointima formation. In colchicine-treated cultures of rat aortic SMCs T-cadherin expression is increased in dividing M-phase cells but decreased in non-dividing cells. Together the data support an association between T-cadherin expression and SMC phenotype.

Angioplasty, Balloon↗

Loss of differences in mesangial cell phenotype between diabetic and normal rats: role of culture passages.

In mesangial cells (MC) isolated from streptozotocin (STZ)-induced diabetic rat kidneys, sensitivity to bradykinin (BK) for the induction of cell division and collagen synthesis, was found to be lower than in normal MC. Nevertheless, decreased activities could be reverted in vitro by insulin, at non-proliferative concentration (Girolami et al (1995), Can J Physiol Pharmacol 73, 848-853). The aim of the present study was to determine whether differences in the properties of diabetic MC could be ascribed to the diabetic state per se, and/or to experimental conditions, ie culture replating. Through successive cell replating, normal and diabetic types of MC were compared in terms of proliferation, contraction, free calcium concentration in response to KCl depolarization, and in relation to the expression of two cytoskeleton proteins specific to muscle cells, myosin and dystrophin. Studies of proliferation, contraction and free calcium concentration consistently showed that passage 5 was a limit beyond which differences between the two MC types were very small and sometimes non-significant. We found that the mean maximum contraction (MMC) and especially the proportion of contractile cells (PCC) among diabetic cells was lower than in normal MC. In addition, loss in proliferation activity and in [Ca2+]i concentrations were also found to occur during these five early passages. Dystrophin, a new marker of contractile phenotype recently described in smooth muscle cell (Leis et al (1994) Cell Biol Toxicol 10, 305), was first localized in MC and was compared with myosin also expressed in MC. However, during the course of cell replating and/or with the diabetic state, no visible quantitative changes were detected in the expression of the two contractile proteins. We conclude that cultured mesangial cells undergo phenotype modulations, as observed in other cells, in particular smooth muscle cells and consequently, comparative studies between normal and diabetic MC should not be carried out after the 5th passage.

Animals↗

Corneal keratocytes: phenotypic and species differences in abundant protein expression and in vitro light-scattering.

PURPOSE: Previous studies suggest that corneal haze after injury involves changes in the light-scattering properties of keratocytes that are possibly linked to the abundant expression of water-soluble proteins. The purpose of this study was to determine the protein expression pattern of keratocytes from different species and different cultured rabbit keratocyte phenotypes and to assess differences in light-scattering in vitro. METHODS: Water-soluble proteins were isolated from corneal epithelial cells and keratocytes of several species, including human (Hu), mouse (Mo), rabbit (Ra), chicken (Ch), and pig (P) and different cultured rabbit keratocyte phenotypes. Proteins were then characterized by SDS-PAGE, tryptic peptide sequence analysis, and Western blot analysis. Light-scattering and actin organization from cultured cells were determined with confocal reflectance and fluorescence microscopy, respectively. RESULTS: Protein expression patterns varied substantially between species and cell types, with five new abundantly expressed proteins identified including, LDH (Ra, Ch), G3PDH (Hu, Ch), pyruvate kinase (Ch), Annexin II (Ch), and protein disulfide isomerase (Ch). Different rabbit keratocyte phenotypes also showed different levels of expression of ALDH1A1 and TKT, with myofibroblasts showing the greatest reduction. Myofibroblasts showed significantly greater (P < 0.05) light-scattering but also showed the greatest organization of actin filaments. CONCLUSIONS: Abundant protein expression is a characteristic feature of corneal keratocytes that is lost when cells are phenotypically modulated in culture. Greater light-scattering by myofibroblasts also provides support for a link between cellular transparency and haze after injury that is possibly related to loss of protein expression or development of prominent actin filament bundles.

Animals↗

Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers.

It has been suggested that perisinusoidal liver cells (PSC) play a pivotal role in the pathogenesis of fibrocontractive changes. Using light and electron microscopic immunolocalization techniques, a series of 207 normal and pathologic human liver specimens were evaluated for the expression of alpha smooth muscle (SM) actin and desmin in this and other nonparenchymal cell types. In normal adult liver tissue, PSCs were practically devoid of desmin and exceptionally stained for alpha-SM actin, whereas this actin isoform frequently was encountered in PSCs from the embryonic to the adolescent period. A broad spectrum of pathologic conditions was accompanied by the presence of alpha-SM actin containing PSCs; these were detected preferentially in periportal or perivenular zones according to the predominant location of the underlying hepatocellular damage. The occurrence of this PSC phenotype generally was associated with fibrogenesis and was in some cases detected earlier than overt collagen accumulation. Fibrous bands subdividing liver tissue in cirrhosis and focal nodular hyperplasia, as well as desmoplastic reaction to malignant tumors, contained alpha-SM actin-rich cells admixed with variable proportions of cells coexpressing desmin. In end stages, this population was less numerous than in active fibrotic or cirrhotic processes. Using immunogold electron microscopy, alpha-SM actin was localized in microfilament bundles of typical PSCs. Our results are compatible with the assumption that the appearance of alpha-SM actin and desmin-expressing myofibroblasts results at least in part from a phenotypic modulation of PSCs.

Actins↗

Platelet-derived growth factor-BB and Ets-1 transcription factor negatively regulate transcription of multiple smooth muscle cell differentiation marker genes.

Platelet-derived growth factor (PDGF)-BB, a potent mitogen for mesenchymal cells, also downregulates expression of multiple smooth muscle (SM) cell (SMC)-specific markers. However, there is conflicting evidence whether PDGF-BB represses SMC marker expression at a transcriptional or posttranscriptional level, and little is known regarding the mechanisms responsible for these effects. Results of the present studies provide clear evidence that PDGF-BB treatment strongly repressed SM alpha-actin, SM myosin heavy chain (MHC), and SM22alpha promoters in SMCs. Of major significance for resolving previous controversies in the field, we found PDGF-BB-induced repression of SMC marker gene promoters in subconfluent, but not postconfluent, cultures. Treatment of postconfluent SMCs with a tyrosine phosphatase inhibitor restored PDGF-BB-induced repression, whereas treatment of subconfluent SMCs with a tyrosine kinase blocker abolished PDGF-BB-induced repression, suggesting that a tyrosine phosphorylation event mediates cell density-dependent effects. On the basis of previous observations that Ets-1 transcription factor is upregulated within phenotypically modulated neointimal SMCs, we tested whether Ets-1 would repress SMC marker expression. Consistent with this hypothesis, results of cotransfection experiments indicated that Ets-1 overexpression reduced transcriptional activity of SMC marker promoter constructs in SMCs, whereas it increased activity of SM alpha-actin promoter in endothelial cells. PDGF-BB treatment increased expression of Ets-1 in cultured SMCs, and SM alpha-actin mRNA expression was reduced in multiple independent clones of SMCs stably transfected with an Ets-1-overexpressing construct. Taken together, results of these experiments provide novel insights regarding possible mechanisms whereby PDGF-BB and Ets-1 may contribute to SMC phenotypic switching associated with vascular injury.

Actins↗

Effects of monohydroxylated fatty acids on arterial smooth muscle cell properties.

The hydroxylated derivatives of polyunsaturated fatty acids may be potent modulators of basic biological responses involved in pathological processes, including atherosclerosis. The object of the present investigation was to study the effects of monohydroxylated fatty acids (namely 12-HETE) on the properties of aortic smooth muscle cells (SMC) in culture. The changes in cell expression of differentiation antigen alpha-SM actin and 2P1A2 was followed by computerized morphometry, using specific monoclonal antibodies and the activation of cells by measuring cell motility. In addition, intracellular [Ca2+]i mobilization and IP3 formation were studied. Finally, the metabolic routes of monohydroxylated compounds and their effects on PGI2 secretion were reported. The results demonstrate that 12-HETE is able to stimulate the phenotypic modulation. PGI2 production and motility of arterial SMCs, despite any detectable activity in increasing [Ca2+]i or IP3 formation. By contrast with parent compounds 15-HETE and 13-HODE, which appear as potent prodifferentiating molecules, 12-HETE is specifically metabolized via a 10-11 reductase pathway in addition to the classical beta-oxidation pathway. Taken together, our results suggest that cellular metabolism of 12-HETE, produced by platelets in the vicinity of the arterial intima, and also by cells present inside the atherosclerotic intima, or associated with modified LDL may play a key role in the atherosclerotic process.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

Expression of exogenous or endogenous green fluorescent protein in adipose tissue-derived stromal cells during chondrogenic differentiation.

Pluripotent stem cells within the adipose stromal compartment, termed adipose-derived stromal cells (ASCs), have the potential to differentiate into a variety of cell lineages both in vitro and in vivo. Imaging with expression of exogenous or endogenous green fluorescent protein (GFP) reporters facilitates the detailed research on ASCs' physiological behavior during differentiation in vivo. This study was aimed to confirm whether ASCs expressing GFP still could be induced to chondrogenesis, and to compare the expression of exogenous or endogenous GFP in ASCs during chondrogenic differentiation. ASCs were harvested from inguinal fat pads of normal nude mice or GFP transgenic mice. Monolayer cultures of ASCs from normal mice were passaged three times and then infected with replication-incompetent adenoviral vectors carrying GFP genes. Allowed to recover for 5 days, Ad/GFP infected ASCs were transferred to chondrogenic medium as well as the ASCs from transgenic mice cultured in vitro over the same passages. The level of GFP in transgenic ASCs maintained stable till 3 months after chondrogenic induction. Whereas, high level of GFP expression in Ad/GFP infected ASCs could last for only 8 weeks and then declined stepwise. Important cartilaginous molecules such as SOX9, collagen type I, collagen type II, aggrecan, collagen type X were assessed using immunocytochemistry, RT-PCR, and Western Blot. The results indicated that no matter the GFP was exogenous or endogenous, it did not influence the chondrogenic potential of ASCs in comparison with the normal controls. Moreover, chondrogenic lineages from ASCs also underwent phenotypic modulation called dedifferentiation as a result of long-term culture in monolayers similar to normal chondrocytes.

Adipose Tissue↗

Role of Rho-kinase in maintaining airway smooth muscle contractile phenotype.

This study aims to investigate the role of Rho-kinase in phenotype switching and proliferation of bovine tracheal smooth muscle. To induce different phenotypic states, bovine tracheal smooth muscle strips were cultured (8 days) in 10% foetal bovine serum (foetal bovine serum, less contractile phenotype) or insulin (1 microM, hypercontractile phenotype) and compared to strips cultured in serum-free medium. In contraction experiments, the Rho-kinase inhibitor (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide (Y-27632, 1 microM) decreased sensitivity to methacholine and KCl and lowered maximal responsiveness to KCl in all strips irrespective of the phenotype present. To investigate the effects of Rho-kinase bovine tracheal smooth muscle phenotypic regulation, strips were pretreated with Y-27632 (1 microM) for 8 days. This resulted in a decreased maximal contractility to both methacholine and KCl, quantitatively comparable to the decrease in contractility induced by platelet-derived growth factor (PDGF, 10 ng/ml). The combination of Y-27632 and PDGF responded additively. Y-27632 did not affect basal or PDGF-induced bovine tracheal smooth muscle cell proliferation, determined both as increases in [3H]thymidine incorporation and cell number. Inhibitors of the p42/p44 mitogen-activated protein kinase (MAPK) pathway, the p38 MAPK pathway and the phosphatidyl inositol (PI) 3-kinase pathway all inhibited PDGF-induced proliferation and phenotype changes. These results show that the functional contribution of Rho-kinase to bovine tracheal smooth muscle contraction is not dependent on phenotypic state. In addition, Rho-kinase is not involved in phenotypic modulation or proliferation induced by PDGF, whereas p42/p44 MAPK, p38 MAPK and PI 3-kinase are. Rho-kinase is, however, a major regulator involved in the basal maintenance of contractility in bovine tracheal smooth muscle.

Animals↗

Heterogeneity of smooth muscle cell populations cultured from pig coronary artery.

OBJECTIVE: Heterogeneous smooth muscle cell (SMC) populations have been described in the arteries of several species. We have investigated whether SMC heterogeneity is present in the porcine coronary artery, which is widely used as a model of restenosis. METHODS AND RESULTS: By using 2 isolation methods, distinct medial populations were identified: spindle-shaped SMCs (S-SMCs) after enzymatic digestion, with a "hill-and-valley" growth pattern, and rhomboid SMCs (R-SMCs) after explantation, which grow as a monolayer. Moreover, the intimal thickening that was induced after stent implantation yielded a large proportion of R-SMCs. R-SMCs exhibited high proliferative and migratory activities and high urokinase activity and were poorly differentiated compared with S-SMCs. Heparin and transforming growth factor-beta2 inhibited proliferation and increased differentiation in both populations, whereas fibroblast growth factor-2 and platelet-derived growth factor-BB had the opposite effect. In addition, S-SMCs treated with fibroblast growth factor-2 or platelet-derived growth factor-BB or placed in coculture with coronary artery endothelial cells acquired a rhomboid phenotype. This change was reversible and was also observed with S-SMC clones, suggesting that it depends on phenotypic modulation rather than on selection. CONCLUSIONS: Our results show that 2 distinct SMC subpopulations can be recovered from the pig coronary artery media. The study of these subpopulations will be useful for understanding the mechanisms of restenosis.

Animals↗

Strain reorganizes focal adhesions and cytoskeleton in cultured airway smooth muscle cells.

Abnormal mechanical stress on pulmonary structures is associated with increased airway resistance and impaired gas exchange as a result of increased airway smooth muscle (ASM) deposition. Using an in vitro system with cultured ASM cells, we have demonstrated that cyclic deformational strain increases ASM cellular myosin and myosin light chain kinase. To determine if these contractile protein increases were accompanied by ultrastructural changes in cells indicating phenotypic modulation, cells subjected to strain were compared to cells grown under static conditions by transmission electron microscopy (TEM) and fluorescent staining. The strained ASM cells oriented perpendicular to the strain direction were more elongated and contained more actin stress fibers than identical cells grown under physically static conditions. The stress fiber bundles were thicker and reorganized parallel to the long axis of the cell. Marked increases in the numbers and lengths of focal adhesions between the cell membrane and the substratum were found by both TEM and immunostaining for talin. Mechanical strain thus increases organization of cytoskeletal elements in cultured ASM cells. Similar effects in vivo may serve to promote the expression of the contractile phenotype of cultured ASM cells independent of other in vivo factors and alter cell contractility. Increased organization of cytoskeletal elements might also increase the efficiency of signal transduction from the extracellular matrix into the cell interior.

Actins↗

Importance of the phenotypic state of vascular smooth muscle cells on the binding and the mitogenic activity of endothelin.

Smooth muscle cells of the rabbit aorta, when grown in vitro, express distinguishable forms of phenotypes (contractile and synthetic). On contractile cells, ET-1 specifically bound to a single class of high affinity (KD = 128 pM) and high capacity (Bmax = 66,000 sites/cell) binding sites. But, whereas affinity of [125I]-ET-1 was not significantly affected by phenotypic modulation, synthetic cells displayed a 10-fold lower [125I]-ET-1 binding capacity than contractile smooth muscle cells. Similarly, the mitogenic effect of ET-1 on smooth muscle cells was considerably lower for synthetic than for contractile cells. The ET-1 receptor on primary cells was recognized by sarafotoxin S6b and the different ET-related peptides with an order of potency [ET-1 greater than S6b greater than ET-3 greater than Big ET-1 much greater than ET(16-21)] identical to that inducing smooth muscle cell growth. Therefore, these data indicate that the binding and the mitogenic effects of ET-1 on smooth muscle cells might be of different magnitudes depending on the phenotypic state of these cells.

Animals↗

Hypoxia, leukocytes, and the pulmonary circulation.

Data are rapidly accumulating in support of the idea that circulating monocytes and/or mononuclear fibrocytes are recruited to the pulmonary circulation of chronically hypoxic animals and that these cells play an important role in the pulmonary hypertensive process. Hypoxic induction of monocyte chemoattractant protein-1, stromal cell-derived factor-1, vascular endothelial growth factor-A, endothelin-1, and tumor growth factor-beta(1) in pulmonary vessel wall cells, either directly or indirectly via signals from hypoxic lung epithelial cells, may be a critical first step in the recruitment of circulating leukocytes to the pulmonary circulation. In addition, hypoxic stress appears to induce release of increased numbers of monocytic progenitor cells from the bone marrow, and these cells may have upregulated expression of receptors for the chemokines produced by the lung circulation, which thus facilitates their specific recruitment to the pulmonary site. Once present, macrophages/fibrocytes may exert paracrine effects on resident pulmonary vessel wall cells stimulating proliferation, phenotypic modulation, and migration of resident fibroblasts and smooth muscle cells. They may also contribute directly to the remodeling process through increased production of collagen and/or differentiation into myofibroblasts. In addition, they could play a critical role in initiating and/or supporting neovascularization of the pulmonary artery vasa vasorum. The expanded vasa network may then act as a conduit for further delivery of circulating mononuclear cells to the pulmonary arterial wall, creating a feedforward loop of pathological remodeling. Future studies will need to determine the mechanisms that selectively induce leukocyte/fibrocyte recruitment to the lung circulation under hypoxic conditions, their direct role in the remodeling process via production of extracellular matrix and/or differentiation into myofibroblasts, their impact on the phenotype of resident smooth muscle cells and adventitial fibroblasts, and their role in the neovascularization observed in hypoxic pulmonary hypertension.

Animals↗

Dedifferentiated chondrocytes cultured in alginate beads: restoration of the differentiated phenotype and of the metabolic responses to interleukin-1beta.

Chondrocytes cultivated in monolayer rapidly divide and lose their morphological and biochemical characteristics, whereas they maintain their phenotype for long periods of time when they are cultivated in alginate beads. Because cartilage has a low cellularity and is difficult to obtain in large quantities, the number of available cells often becomes a limiting factor in studies of chondrocyte biology. Therefore, we explored the possibility of restoring the differentiated properties of chondrocytes by cultivating them in alginate beads after two multiplication passages in monolayer. This resulted in the reexpression of the two main markers of differentiated chondrocytes: Aggrecan and type II collagen gene expression was strongly reinduced from day 4 after alginate inclusion and paralleled protein expression. However, 2 weeks were necessary for total suppression of type I and III collagen synthesis, indicators of a modulated phenotype. Interleukin-1beta, a cytokine that is present in the synovial fluid of rheumatoid arthritis patients, induces many metabolic changes on the chondrocyte biology. Compared with cells in primary culture, the production of nitric oxide and 92-kDa gelatinase in response to interleukin-1beta was impaired in cells at passage 2 in monolayer but was fully recovered after their culture in alginate beads for 2 weeks. This suggests that the effects of interleukin-1beta on cartilage depend on the differentiation state of chondrocytes. This makes the culture in alginate beads a relevant model for the study of chondrocyte biology in the presence of interleukin-1beta and other mediators of cartilage destruction in rheumatoid arthritis and osteoarthrosis.

Aggrecans↗

Expression of PDGF alpha- and beta-receptors in rat arterial smooth muscle cells is phenotype and growth state dependent.

Adult rat arterial smooth muscle cells were shown to express mRNA for the platelet-derived growth factor (PDGF) alpha- and beta-receptors and to bind radioiodinated PDGF-AA and PDGF-BB in a phenotype-dependent and growth state-dependent manner. PDGF alpha-receptor mRNA was not detected in the intact aortic media, but appeared as the cells converted from a contractile to a synthetic phenotype during serum-free primary culture. PDGF beta-receptor mRNA was expressed already in vivo, and increased further as the cells were isolated and cultured in vitro. Exposure of the cells to human platelet PDGF resulted in increased PDGF alpha-receptor mRNA levels, decreased PDGF beta-receptor mRNA levels, and decreased binding of both PDGF-AA and PDGF-BB. Following removal of the exogenous mitogen, the content of PDGF alpha- and beta-receptor mRNA increased, as did the binding of PDGF-AA and PDGF-BB. Subsequently, the content of PDGF A-chain mRNA started to rise, and the cells retained a high rate of DNA synthesis in a serum-free medium. As a result of this autocrine stimulation, the PDGF receptors were down-regulated. Although smooth muscle cells in serum-free primary cultures bound the different PDGF isoforms to a varying extent (AA less than AB less than BB), the replicative response was of a similar magnitude. Subcultured cells bound the different PDGF isoforms in similar proportions as the primary cells. Contrary to the situation in primary cells, there was a direct correlation between the binding level and the DNA synthetic response. Moreover, the subcultured cells did not replicate in a serum-free medium. These observations support the idea that the phenotypic modulation of arterial smooth muscle cells in primary culture prepares the cells to activate autocrine growth mechanisms. When stimulated with an exogenous mitogen, they enter the cell cycle and are thereafter able to stimulate their own growth in an autocrine manner by production of PDGF-AA or a closely related molecule.

Animals↗

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.

Animals↗

Differentiation of rat oval cells after activation of peroxisome proliferator-activated receptor alpha43.

Peroxisome proliferators (PPs) act as nongenotoxic tumor promoters in rodents. Their hepatocarcinogenicity requires the presence of the PP-activated receptor alpha (PPARalpha); however, the exact role played by this transcription factor in the liver, more precisely in liver cell growth and differentiation, is not known. The aim of this study was to investigate the role of PPARalpha in oval cells, which are considered to be closely related to liver stem cells, act as bipotential progenitors for the two main hepatic lineages, and have been implicated as playing a role in several models of liver carcinogenesis. We studied the PPARalpha-mediated response of primary oval cells isolated from rats fed a choline-deficient ethionine-supplemented diet (CDE diet, a regimen commonly used for the induction of oval cell proliferation in rodents) with or without cotreatment with WY14,643, a prototype PPARalpha-activator. PPARalpha was expressed at relatively low levels in primary oval cells from rats fed the CDE diet alone. In vivo treatment with WY14,643 for 2-6 weeks induced, in the oval cells, the expression of PPARalpha as well as that of the PPARalpha-responsive genes encoding fatty acyl-CoA oxidase and cytochrome P450 4A1. Moreover, the oval cell response to WY14,643 was accompanied by an overall phenotypic modulation toward the hepatocyte lineage. In addition, the PPARalpha activator induced, among the oval cells, a subpopulation of transitional cells showing features of maturing hepatocytes expressing the oncofetal marker, alpha-fetoprotein. These results show that oval cells are responsive to PPs and strongly argue for a role of PPARalpha in the differentiation/maturation of rat oval cells. In the absence of the CDE diet regimen, 9-week treatment with WY14,643 lead to the appearance of a population of large-sized cells somewhat similar to the transitional cells. However, these cells showed little expression of markers of mature hepatocytes, consistent with a block during their maturation process, i.e., they are resistant to PPARalpha-mediated differentiation. Interestingly, the phenotype of these cells resembled that of the cells usually found in neoplastic foci induced by PPs. Our results, together with previous reports, suggest the involvement of oval cells in the hepatocarcinogenicity of PPs.

Animals↗

Importance of critical metabolites and cellular interactions in the biology of microregions of tumors.

Heterogeneity of cellular microenvironments and associated differences in phenotypic expression of cells in solid tumors occur as a consequence of deficiencies of vascularization. Intervascular microregions of tumors and micrometastases can be modeled by multicellular spheroids in vitro. These develop concentration gradients of critical metabolites such as oxygen, glucose, and probably also other important nutrients, hormones, and growth factors. Drug penetration may also be reduced. Concentrations of these factors decrease from the periphery towards the center, but gradients develop in the opposite direction for certain metabolic and necrotic products such as lactate and pH. Such gradients significantly modify the proliferative status of the cells, viability, clonogenicity, cell cycle distribution, antigen expression, and differentiation. Stress of oxygen and glucose deprivation induces synthesis of a specific set of proteins. Cellular interactions can also modulate phenotypic expression, including oxygen and glucose consumption rates and sensitivity to radiation and drugs. The mechanisms of these are unknown, but changes in cell-cell communication and DNA damage and repair have been demonstrated in spheroids. Microenvironmental and cellular changes may be transient. Thus, the characteristics of cells in tumor microregions may be quite different from the intrinsic cellular properties observed after growth of cells in standard culture systems. These changes may have significant effects on responses to therapeutic agents.

Adenocarcinoma↗