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Results for “Phenotypic modulation”

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PubMed1999

Embryonic smooth muscle myosin heavy chain SMemb is expressed in pressure-overloaded cardiac fibroblasts.

Left ventricular hypertrophy (LVH) is a secondary adaptation to increased external load. Various qualitative and quantitative changes in myocytes and extracellular components occur during the development of LVH. It has recently been demonstrated that alpha-smooth muscle actin (alpha-SMA)-expressing myofibroblasts appear in the interstitium of the heart subjected to increased workload suggesting that cardiac fibroblasts as well as myocytes alter their phenotype in response to pressure overload. In the present study, to explore the load-induced response and phenotypic modulation of cardiac fibroblasts, the localization of embryonic smooth muscle myosin heavy chain (SMemb) and alpha-SMA in thoracic aorta-constricted rat hearts was investigated by immunohistochemistry, and the morphology of the SMemb-expressing cells was examined by electron microscopy. In addition, to clarify the mechanisms by which SMemb is induced in pressure-overloaded hearts, mRNA expression of SMemb in aorta-constricted rat hearts and in transforming growth factor-beta1 (TGF-beta1)-treated or mechanically-stretched cultured cardiac fibroblasts was investigated. Enhanced staining of SMemb and alpha-SMA was detected in the interstitial spindle-shaped cells in the fibrotic lesions of the pressure-overloaded left ventricles by immunohistochemistry. These cells were demonstrated by electron microscopy to have features specific for activated fibroblasts such as serrated nuclei or prominent rough endoplasmic reticulum. These cells also had characteristic features of myofibroblasts, i.e. irregularly arranged actin filaments and scattered dense bodies. Northern blot analysis revealed increased mRNA levels of SMemb both in aorta-constricted rat hearts and in cultured cardiac fibroblasts stimulated by TGF-beta1 or by mechanical stretch. These results suggest that SMemb may be a molecular marker both for the detection of activated cardiac fibroblasts that may play important roles in the remodeling of pressure-overloaded cardiac interstitium, and for the identification of the regu latory mechanisms that control the phenotypic modulation of cardiac fibroblasts in response to pressure overload.

Actins↗
PubMed2003

Regulation of SM22 alpha expression by arginine vasopressin and PDGF-BB in vascular smooth muscle cells.

Vascular smooth muscle (SM) cells (VSMC) undergo phenotypic modulation in vivo and in vitro. This process involves coordinated changes in expression of multiple SM-specific genes. In cultured VSMC, arginine vasopressin (AVP) increases and PDGF decreases expression of SM alpha-actin (SMA), the earliest marker of SM cells (SMC). However, it is unknown whether these agents regulate other SM genes in a similar fashion. SM22 alpha appears secondary to SMA during development and is also a marker for SMC. This study examined the regulation of SM22 alpha expression by AVP and PDGF in cultured VSMC. Levels of SM22 alpha mRNA and protein were increased by AVP and suppressed by PDGF. Consistent with these changes, AVP increased SM22 alpha promoter activity, whereas PDGF inhibited basal promoter activity and blocked AVP-induced increase. Activation of both JNK and p38 MAPK pathways was necessary for AVP-mediated induction of SM22 alpha promoter. Expression of constitutively active Ras produced similar suppressions on SM22 alpha promoter activity as PDGF. Signaling relayed from PDGF/Ras activation involved Raf, or a protein that competes for this site, Ral-GDS, and phosphatidylinositol 3-kinase activation. Truncational analysis showed that the proximal location of three CArG boxes in the promoter was sufficient for AVP stimulation. Mutations in this CArG box reduced basal and AVP-stimulated promoter activity without effecting PDGF suppression. Overexpression of serum response factor enhanced basal and AVP-stimulated promoter activity but had no effect on PDGF-BB-induced suppression. These data indicate that AVP and PDGF initiate specific signaling pathways that control expression of multiple SM genes leading to phenotypic modulation.

Animals↗
PubMed2005

PDGF-induced signaling in proliferating and differentiated vascular smooth muscle: effects of altered intracellular Ca2+ regulation.

OBJECTIVE: Platelet-derived growth factor-BB (PDGF)-induced intracellular signaling is involved in phenotypic modulation of vascular smooth muscle (VSM). This study has examined the PDGF-induced Ca2+ increase and the resultant effect on signaling pathways in proliferative compared with fully differentiated VSM. METHODS: PDGF-induced changes in Ca2+ were measured in portal vein (PV) myocytes from 2-4-day-old (proliferating), compared to 6-week-old (differentiated), Sprague Dawley rats. Phospholipase C (PLC)gamma expression and activation of extracellular signal-regulated kinase (ERK) 1/2 was determined by immunoblotting or confocal immunolabelling. Activation of the Ca(2+)-dependent transcription factor, nuclear factor of activated T-cells (NFATc), was assessed by electromobility shift assay. RESULTS: PDGF increased the intracellular Ca2+ concentration in differentiated, but not in proliferating, PV myocytes. This is probably due to very low expression of PLC(gamma) in proliferating PV. In 6-week-old PV, PDGF stimulation induced nuclear translocation and activation of NFATc. PDGF did not induce NFATc activation in neonatal PV. PDGF-induced ERK1/2 activation was observed in both 2-4-day-old and 6-week-old PV. In 6-week-old PV, ERK1/2 activation was Ca(2+)-dependent and protein kinase C-dependent. However in 2-4-day-old PV, PDGF-induced ERK1/2 activation was via a Ca(2+)-independent, atypical protein kinase C. PLC(gamma) expression was also decreased in the neointima, compared to media, of balloon-injured rabbit subclavian arteries. CONCLUSIONS: The regulation of PDGF-induced Ca2+ increases by PLC(gamma) expression in VSM may provide a mechanism for coordinating different signaling pathways leading to activation of specific transcription factors. This may play an important role in the phenotypic modulation of VSM.

Animals↗
PubMed2000

Modulation of phenotype and induction of irregular vessels accompany high tumorigenic potential of clonal human glioma cells xenografted to nude-rat brain.

Three phenotypically different clonal human glioma cell lines were injected stereotactically into nude-rat brains, to determine their individual growth potential and to establish an in vivo system in which different therapeutic modalities could be tested. As assessed by serial sectioning, microscopic evaluation, and computer analysis, the mean approximate tumour volume after 3-7 weeks in vivo was 0.42 mm(3) for U-343 MG, 2.6 mm(3) for U-343 MGa Cl2:6, and 50.3 mm(3) for U-343 MGa 31L. When compared with the initial injected cell volume, only U-343 MGa 31L had increased in size, U-343 MGa Cl2:6 remained approximately the same but showed a certain proliferative potential, and U-343 MG regressed. Thus, only U-343 MGa 31L cells had high tumorigenic potential, invaded and replaced brain tissue in every direction, while U-343 MGa Cl2:6 cells grew in sheet-like tumour extensions along white-matter nerve-fibre tracts, in this respect mimicking foetal astrocytes. The tumorigenic potential of the U-343 MGa 31L cell clone was associated with a variable phenotype, as observed when the in vivo and in vitro characteristics were compared. The in vivo phenotype was characterized by the loss of GFAP immunoreactivity, the gain of heterogeneously distributed cellular tenascin, fibronectin, and laminin, but absence of extracellularly deposited material, and by the formation of irregular vessels. It appears that the intrinsic capacity of glioma cells to adapt to in vivo conditions is decisive for their tumorigenicity in the brain, rather than any single phenotypic property in itself. Moreover, the 2 glioma cell clones best suited for in vitro growth were no longer tumorigenic.

Animals↗
PubMed2004

Rho expression and activation in vascular smooth muscle cells.

Phenotypic modulation of smooth muscle cells (SMC) involves dramatic changes in expression and organization of contractile and cytoskeletal proteins, but little is known of how this process is regulated. The present study used a cell culture model to investigate the possible involvement of RhoA, a known regulator of the actin cytoskeleton. In rabbit aortic SMC seeded into primary culture at moderate density, Rho activation was high at two functionally distinct time-points, first as cells modulated to the "synthetic" phenotype, and again upon confluence and return to the "contractile" phenotype. Rho expression increased with time, such that maximal expression occurred upon return to the contractile state. Transient transfection of synthetic state cells with constitutively active RhoA (Val14RhoA) caused a reduction in cell size and reorganization of cytoskeletal proteins to resemble that of the contractile phenotype. Actin and myosin filaments were tightly packed and highly organised while vimentin localised to the perinuclear region; focal adhesions were enlarged and concentrated at the cell periphery. Conversely, inhibition of endogenous Rho by C3 exoenzyme resulted in complete loss of contractile filaments without affecting vimentin distribution; focal adhesions were reduced in size and number. Treatment of synthetic state SMC with known regulators of SMC phenotype, heparin and thrombin, caused a modest increase in Rho activation. Long-term confluence and serum deprivation induced cells to return to a more contractile phenotype and this was augmented by heparin and thrombin. The results implicate RhoA for a role in regulating SMC phenotype and further show that activation of Rho by heparin and thrombin correlates with the ability of these factors to promote the contractile phenotype.

Animals↗
PubMed2004

Modulation of phenotype of human prostatic stromal cells by transforming growth factor-betas.

BACKGROUND: We investigated the effects of transforming growth factor (TGF)-betas on morphological and receptor phenotypes, as well as proliferation of four currently established human prostatic myofibroblast cell lines and one commercially available prostatic stromal cell line. METHODS: The effects of TGF-betas on morphological changes and proliferation of the cells were studied by immunohistochemistry and bromodeoxyuridine assay, respectively. The expression of alpha 1-receptor subtypes was measured by real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and the radioligand binding assay for the receptors was also performed. RESULTS: TGF-betas 1, 2, and 3 induced expression of desmin and myosin of cells of the established cell lines, and significantly inhibited their growth. The alpha 1a-receptor was expressed only in the commercially available cell line and alpha 1b and 1d, in all cell lines. TGF-beta 1 suppressed the expression of all three subtypes of the alpha 1-receptor. The binding sites of cells of all the cell lines were reduced by treatment with this growth factor. CONCLUSIONS: TGF-betas may induce human prostatic stromal cells to express the smooth muscle phenotype and inhibited their growth. However, the growth factor reduced the binding sites of the receptor and suppressed mRNA expression of its subtypes, suggesting that morphological and receptor phenotypes may be regulated via more than one pathway by TGF-beta(s).

Cell Division↗
PubMed1997

Fibroblasts can modulate the phenotype of malignant epithelial cells in vitro.

An organotypic, tridimensional cell culture, also called a raft system, was used to study the influence of fibroblasts on epithelial carcinogenesis in a cell line derived from laryngeal squamous cell carcinoma and harboring a mutated p53. Differences between the effects of normal fibroblasts and those of tumor-derived fibroblasts were compared by means of fibroblasts taken from the normal skin and from the tumor of a cancer patient and cultivated with epithelial carcinoma cells in an organotypic culture. To study cell contact-mediated changes, the fibroblasts were either simply embedded in collagen matrix or additionally brought into direct contact with epithelial cells. Control epithelial cells were cultivated without any fibroblasts in an organotypic model. A protein panel [p53, p21, PCNA, bcl-2, Ki67, total cytokeratin (CK), CK 8, CK 10, CK 17, CK 18, CK 19, vimentin] involved in cell cycling and epithelial differentiation was assessed immunocytochemically in all organotypic cultures with fibroblasts, in tumor cells cultivated as a monolayer, and in the original tumor sample. The most dysplastic phenotype was obtained when tumor-derived fibroblasts were used in direct contact with epithelial cells, whereas the most benign phenotype was seen when skin fibroblasts had no contact with them. The intensive staining seen for p53 can be explained by p53 mutations also reflecting the weak expression of p21 and abundant expression of PCNA. The intensive Ki67 staining seen in all sections paralleled that of PCNA and marked active cellular proliferation. The CK staining pattern seen in cultured epithelia toward embryonic CKs, CK 8 and CK 18, suggested a simple epithelial phenotype. CK 19 was found only in the epithelium where no direct contacts had occurred. Vimentin expression increased when the raft epithelium was shifting toward a more benign phenotype. The results stress the importance of the origin of fibroblasts as well as the role of direct cellular contacts in modifying the epithelial phenotype even when the epithelial cells are malignant.

Adult↗
PubMed1997

Beta 2 laminins modulate neuronal phenotype in the rat retina.

The production of cell types in the vertebrate retina follows a stereotyped time course. We have focused on a component of the extracellular matrix that may guide this schedule: the laminin beta 2 chain. Here, we have asked directly whether heterotrimeric laminins containing the laminin beta 2 chain can promote the production of presumptive rod photoreceptors ("rods") and have correlated changes in rod production with changes in the production of other cell types. In cultures in which few rods, but many Müller and bipolar cells, are produced, the production of rods can be enhanced sixfold and that of bipolar cells can be reduced by 66%, by exposing cells to a laminin beta 2-rich matrix. Substitution of a laminin beta 2-depleted matrix (created with antisense RNA) returns the density of rods and bipolar cells to control levels. These linked alterations in phenotype expression suggest that laminins may control the choice between rod photoreceptor and rod bipolar cell fates.

Animals↗
PubMed2002

TGFbeta1 modulates the phenotype of Schwann cells at the transcriptional level.

We have examined the effects of transforming growth factor beta1 (TGFbeta1) on gene expression in cultured rat Schwann cells (SCs). TGFbeta1 decreased the steady-state mRNA levels of several genes that are expressed by myelinating SCs but had varied effects on the mRNA levels of NCAM, L1, GAP-43, and p75-genes that are expressed by denervated and nonmyelinating SCs. TGFbeta1 antagonized the effects of forskolin on the mRNA levels of the transcription factors Oct-6/tst-1/SCIP and Krox20. Transcriptional run-off analysis demonstrated that the effects of TGFbeta1 on gene expression occur at least in part at the level of transcription. Thus, TGFbeta1 suppresses the expression of genes that characterize the different phenotypes of SCs, and these changes occur at least in part at a transcriptional level.

Animals↗
PubMed1987

Rat brain microvessel extracellular matrix modulates the phenotype of cultured rat type 1 astroglia.

Astroglia from immature rat cerebral white matter which were plated on the insoluble extracellular matrix (ECM) secreted by rat cerebral microvessel endothelial cells (RCMEC) and maintained in a defined medium were induced to become stellate and to express glutamine synthetase. These effects were not elicited by RCMEC-conditioned medium. ECM secreted by rat pleural mesothelium elicited a lesser proportion of stellate astroglia and did not induce glutamine synthetase.

Animals↗
PubMed1992

Directional atherectomy for treatment of restenosis within coronary stents: clinical, angiographic and histologic results.

OBJECTIVES: The safety and long-term results of directional coronary atherectomy in stented coronary arteries were determined. In addition, tissue studies were performed to characterize the development of restenosis. METHODS: Directional coronary atherectomy was performed in restenosed stents in nine patients (10 procedures) 82 to 1,179 days after stenting. The tissue was assessed for histologic features of restenosis, smooth muscle cell phenotype, markers of cell proliferation and cell density. A control (no stenting) group consisted of 13 patients treated with directional coronary atherectomy for restenosis 14 to 597 days after coronary angioplasty, directional coronary atherectomy or laser intervention. RESULTS: Directional coronary atherectomy procedures within the stent were technically successful with results similar to those of the initial stenting procedure (2.31 +/- 0.38 vs. 2.44 +/- 0.35 mm). Of five patients with angiographic follow-up, three had restenosis requiring reintervention (surgery in two and repeat atherectomy followed by laser angioplasty in one). Intimal hyperplasia was identified in 80% of specimens after stenting and in 77% after coronary angioplasty or atherectomy. In three patients with stenting, 70% to 76% of the intimal cells showed morphologic features of a contractile phenotype by electron microscopy 47 to 185 days after coronary intervention. Evidence of ongoing proliferation (proliferating cell nuclear antigen antibody studies) was absent in all specimens studied. Although wide individual variability was present in the maximal cell density of the intimal hyperplasia, there was a trend toward a reduction in cell density over time. CONCLUSIONS: Although atherectomy is feasible for the treatment of restenosis in stented coronary arteries and initial results are excellent, recurrence of restenosis is common. Intimal hyperplasia is a nonspecific response to injury regardless of the device used and accounts for about 80% of cases of restenosis. Smooth muscle cell proliferation and phenotypic modulation toward a contractile phenotype are early events and largely completed by the time of clinical presentation of restenosis. Restenotic lesions may be predominantly cellular, matrix or a combination at a particular time after a coronary procedure.

Actins↗
PubMed2004

Gender modulates cardiac phenotype development in genetically modified mice.

Recent research using genetically modified mice has revealed significant sex differences in cardiac phenotypes. In the majority of strains, females display a lower mortality, less severe hypertrophy, better preserved function and mitigated cardiac pathology compared with male counterparts. Thus, gender is an independent determinant for the development of cardiac phenotype in murine models. While there is strong evidence for estrogen as a cardiac protector, emerging evidence indicates adverse actions of testicular hormones that might be responsible in part for the sex differences. Studies using mouse models have also revealed novel information on signalling mechanisms mediating the sex difference.

Animals↗
PubMed2005

Parenteral lipids modulate leukocyte phenotypes in whole blood, depending on their fatty acid composition.

To characterize the immunological effects of various lipids that are applied as part of total parenteral nutrition (TPN) formulations, we analyzed phenotypical changes in leukocytes following lipid exposure. Importantly, the study was performed with whole blood in order to prevent the functional changes that are induced by isolation procedures. Briefly, blood samples from 10 healthy volunteers were incubated with lipids containing pure long-chain triglycerides (L), mixed long- and medium-chain triglycerides (LM), synthetic structured lipids (SL), or emulsions based on olive oil (OO), or fish oil (FO). After immune fluorescent staining, leukocyte phenotype characteristics were analyzed by flowcytometry. Exposure to LM increased in a dose-dependent manner the expression of membrane surface markers for adhesion (CD11b) and degranulation (CD66b), while decreasing CD62L, on neutrophils and monocytes. These findings demonstrate that LM activates leukocytes in peripheral whole blood. On the other hand, decreased expression of activation markers was observed with L and FO. Lipids effects on the phenotype of T lymphocytes and Natural Killer cells were not seen during incubation for up to 4 h. These results indicate that (i) the composition of TPN formulations with regard to lipid structure has implications for the function of exposed immune competent cells and (ii) medium-chain triglycerides, which have been regarded as functionally inert deliverers of fuel calories, have distinct biological effects.

Adult↗
PubMed2002

Lochial secretions of Escherichia coli- or Arcanobacterium pyogenes-infected bovine uteri modulate the phenotype and the functional capacity of neutrophilic granulocytes.

It has been suggested that in cases of puerperal endometritis of cattle infected with Escherichia coli and Arcanobacterium pyogenes, the neutrophils are compromised in their defense capacity or downregulated functionally. In addition to direct bacterial effects, contents of lochial secretions and secreted products of locally activated polymorphonuclear neutrophilic granulocytes (PMNs) may also account for changes in function of freshly immigrating neutrophils. In this study, lochial secretions were obtained from healthy cows and from cows infected by E. coli or A. pyogenes. Separated uterine PMN of infected cows displayed an altered phenotype and function which correlated with the degree of bacterial contamination. Concurrently tested circulating PMN showed no such changes. Infected lochial secretions sterilized by filtration also changed the phenotype of blood PMN. Lochial secretions of healthy cows displayed only minor effects. The effects on PMN function in infected cows varied: ingestion was less affected, whereas generation of reactive oxygen species (ROS) was severely depressed. Concurrently tested purified bacterial products (solubles and fragments) of E. coli and A. pyogenes did not induce the phenotypical and functional changes observed in blood PMN. Since infected lochia also contained high numbers of immigrated and probably activated PMN, the influence of supernatants from phorbol myristate acetate-activated PMN were tested on freshly isolated blood PMN. Such supernatants also increased the expression of certain surface molecules and inhibited the ROS generation. Thus, reduced function and altered phenotypes of PMN which immigrate into the uteri of cows with bacterial endometritis is due not only to interactions with bacteria or bacterial products, but is also to the uterine milieu.

Actinomycetaceae↗
PubMed1999

Early hypoxia modulates the phenotype of dopaminergic cells in rat di- and mesencephalic cell cultures and induces a higher vulnerability of non-dopaminergic neurons to a second hypoxic exposure.

To investigate long-term effects of hypoxia on a cellular level, di- and mesencephalic cell cultures were exposed to hypoxia on in vitro day 2 (incubation in culture medium, pO2 = 10-20 mmHg, 24 h) and on in vitro day 13 (incubation in an electrolyte solution, pO2 = 10-20 mmHg, 8 h). The numbers of neuron-specific enolase immuno-reactive (NSE-IR) and tyrosine hydroxylase immuno-reactive (TH-IR) neurons and the levels of dopamine, its main metabolites and the spontaneous and potassium-stimulated DA release were determined on DIV 15. Hypoxia on DIV 2 did not affect the numbers of NSE-IR and TH-IR neurons, but increased the dopamine content and dopamine release by about 100% in both di-and mesencephalic cultures. In addition, this hypoxia increased the vulnerability of non-TH-IR neurons to the second hypoxic episode applied during more advanced stages of the culture development on DIV 13. On the contrary, hypoxia exposure did not affect the vulnerability of TH-IR cells.

Animals↗
PubMedDate not supplied

Modulation of phenotype, cytokine production and stimulatory function of CD34+-derived DC by NiCl(2) and SDS.

One of the most promising alternatives to identify the sensitizing potency of new products is the in vitro culture of human dendritic cells (DC). In vivo, dendritic cells present in the skin are highly specialized antigen-presenting cells (APC) of the immune system, which play a crucial role in the induction of allergic reactions. The DC produce specific cytokines and upregulate specific co-stimulatory molecules in addition to the antigen-MHC complex in order to promote an optimal T-cell activation. The aim of our study is to assess the phenotype, cytokine production and autologous T-cell stimulatory capacity of the in vitro CD34+-derived dendritic cells after 24 hours of incubation with the metal allergen NiCl(2) (100-300 microM) and the irritant sodium dodecyl sulfate (SDS; 0.01%), in order to find a sensitive endpoint to discriminate between sensitizers and irritants. After exposure to Ni, a significant increase in the number of CD83+ and CD86+ cells was noticed. The intensity of CD86 as well as the intensity of the HLA-DR molecule on the DC also showed a significant increase. The expression of the co-stimulatory molecule CD80 was not changed after exposure. SDS was not able to increase the expression of any of the analysed markers. The production of IL-6 increased significantly after exposure of dendritic cells to Ni, but not after SDS exposure. Results on tumor necrosis factor-alpha (TNF-alpha) production are somewhat equivocal. Although not statistically significant, TNF-alpha was upregulated in one out of three experiments after 48 hours of exposure to the Ni allergen, but increases were also noticed after exposure to SDS (two out of three experiments). Both exposure to Ni and SDS caused an upregulation (not significant) in the IL-12 production by DC, but the production was higher in Ni-exposed DC compared to SDS-exposed cells. In none of the exposed DC cultures was it possible to detect IL-1 beta. The antigen-presenting capacity of the DC in autologous MLR could not be demonstrated. Nevertheless, T-cell proliferation after DC stimulation was noticed in allogenic MLR.

Allergens↗
PubMed2001

Cell-cell contacts with epithelial cells modulate the phenotype of human macrophages.

Interactions of macrophages with epithelium represent one of the pathways involved in regulating local immune mechanisms. We studied the effect of cell-cell contact with an epithelial monolayer on the phenotype of macrophages. Human monocytes and THP-1 macrophages were co-cultured with monolayers of human bronchial epithelial cells (HBECs), the alveolar type II-like cell line A549, renal adenocarcinoma epithelial cells (RA), and the lung fibroblast strain HFL-1. The expression of CD11b, CD14, CD54, and HLA-DR was measured by immunocytochemistry and flow cytometry and showed epithelial cell induction of CD54 and HLA-DR in monocytes and of all antigens in THP-1 cells. Co-culture with fibroblasts did not change the phenotype of macrophages. Separation by a filter insert inhibited most of the effects. Culture supernatants did not induce prominent phenotypic changes. Cell-cell contacts with epithelium appear to be of importance in regulating the phenotype of macrophages.

Cell Communication↗