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Results for “Phenotypic modulation”

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At least 487 records · Page 27Linked to original sources

PubMed1994

Hyperplastic growth of aortic smooth muscle cells in renovascular hypertensive rabbits is characterized by the expansion of an immature cell phenotype.

Smooth muscle cells (SMCs) of rabbit aorta undergo marked changes in myosin isoform content during development. Analysis of nonmuscle myosin composition at the protein level has permitted the identification of three phases in the SMC differentiation process: fetal, postnatal, and adult. Using monoclonal antibodies specific for smooth muscle and nonmuscle myosins and extra domain A of fibronectin as well as cDNA probes for platelet-derived growth factors (PDGF) and various procollagens, we have evaluated the differentiation pattern of aortic SMCs in two-kidney, one-clip hypertensive rabbits. Morphometric and bromo-deoxyuridine studies indicate that hypertrophy of aortic media along with intimal thickening occurring in hypertensive animals is due to SMC hyperplasia. Western blotting experiments performed on aortic specimens from hypertensive animals with antimyosin antibodies revealed the appearance of a myosin isoform pattern of the "immature" type. Immunofluorescence tests showed that these cells are localized in the thickened intima or distributed in the underlying media (sparsely or in groups). Similarly, the fibronectin variant showing the extra domain A, peculiar to "phenotypically modulated" SMCs, appeared in intimal thickening, and its expression followed the time course of nonmuscle myosin expression. Counting of postnatal-type SMCs in the aortic media revealed that this cell population increases markedly with hypertension (2- up to 15-fold at 4 months) and then declines to near control level in 8-month hypertensive rabbits. Diminution of postnatal-type SMCs at later stages of hypertension was temporally correlated with the slowing down of aortic wall hypertrophy. Average levels of mRNAs, as determined by densitometric analysis in aortas from 1- and 2.5-month hypertensive rabbits, showed an increased expression for PDGF beta receptor (up to twofold), procollagen type I (alpha 1, threefold), procollagen type III (alpha 1, twofold), and fibronectin (up to threefold) compared with controls. Conversely, the steady-state levels of mRNAs for PDGF (A and B chain), PDGF alpha receptor, TGF-beta 1, and procollagen type IV (alpha 1) did not increase significantly. These results provide evidence that in adult renovascular hypertensive rabbits, the hyperplastic growth of aortic SMCs is accompanied by the expansion of an "immature" cell phenotype characteristic of the early stages of development.

Actins↗

PubMed2000

p53-mediated differentiation of the erythroleukemia cell line K562.

The tumor suppressor gene p53 can mediate both apoptosis and cell cycle arrest. In addition, p53 also influences differentiation. To further characterize the differentiation inducing properties of p53, we overexpressed a temperature-inducible p53 mutant (ptsp53Val135) in the erythroleukemia cell line K562. The results show that wild-type p53 and hemin synergistically induce erythroid differentiation of K562 cells, indicating that p53 plays a role in the molecular regulation of differentiation. However, wild-type p53 did not affect phorbol 12-myristate 13-acetate-dependent appearance of the megakaryocyte-related cell surface antigens CD9 and CD61, suggesting that p53 does not generally affect phenotypic modulation. The cyclin-dependent kinase inhibitor p21, a transcriptional target of p53, halts the cell cycle in G1 and has also been implicated in the regulation of differentiation and apoptosis. However, transiently overexpressed p21 did neither induce differentiation nor affect the cell cycle distribution or viability of K562 cells, suggesting that targets downstream of p53 other than p21 are critical for the p53-mediated differentiation response.

Animals↗

PubMed2001

Ectopic expression of Smad7 inhibits transforming growth factor-beta responses in vascular smooth muscle cells.

Vascular injury stimulates the cytokine-growth factor network in the vascular wall, including transforming growth factor-beta (TGF-beta). Reportedly, the intracellular signaling of TGF-beta is mediated by Smad proteins. We tested the effects of the ectopic expression of inhibitory Smads in cultured rat smooth muscle cells (SMC) to identify the role of TGF-beta/Smad signaling on the phenotypic modulation of SMC. The cells exposed to human recombinant TGF-beta1 (10 ng/ml) were stimulated Smad2 phosphorylation. Infection with the replication-deficient adenovirus vector expressing Smad7, but not bacterial beta-galactosidase or Smad6, was found to inhibit TGF-beta-induced Smad2 phosphorylation in a dose-dependent manner. TGF-beta suppressed the serum-induced proliferation of SMC from 36.3% to 51.0% (p<0.01), as measured by hand-counting, and this inhibition was attenuated by the ectopic expression of Smad7 (from 30.7% to 74.8% of the reduction of TGF-beta-response, p<0.05), but not Smad6. A BrdU incorporation assay also showed that TGF-beta-mediated growth inhibition was attenuated by exogenous Smad7 and that this inhibition can be reversed by an additional expression of exogenous Smad2. TGF-beta increased the expression of alpha-smooth muscle actin and myosin heavy chain by 1.3-fold and 1.6-fold in comparison to the control, respectively, and these increases were attenuated by exogenous Smad7, but not Smad6. Our data indicate that Smads mediate TGF-beta responses on SMC phenotypes. Smad7, but not Smad6, may specifically act as an inhibitor of TGF-beta responses.

Animals↗

PubMed1997

The cell biology of atherosclerosis--new developments.

In the development of atherosclerotic lesions, three basic processes occur: 1) invasion of the artery wall by leucocytes, particularly monocytes and T-lymphocytes; 2) smooth muscle phenotypic modulation, proliferation, and synthesis of extracellular matrix; and 3) intracellular (macrophage and smooth muscle) lipoprotein uptake and lipid accumulation. Invasion of the vessel wall by leucocytes is mediated through the expression of adhesion molecules on both leucocytes and the endothelium making them 'sticky'. The adhesion molecules are induced by inflammatory mediators released from leucocytes and endothelium, and these in turn are induced by high serum cholesterol levels or complement fragments. Leucocytes which have adhered to the endothelium are chemo-attracted into the vessel wall by cytokines produced by early arriving leucocytes or by low density lipoprotein which has passively passed into the wall, in the process being trapped and oxidised. The oxidised low density lipoprotein is taken up by scavenger receptors (which are not subject to down-regulation) on both macrophages and smooth muscle cells. The overaccumulation of lipid is toxic to the cells and they die contributing to the central necrotic core. The macrophages and T-lymphocytes produce substances which induce smooth muscle cells of the artery wall to change from a 'contractile' (high volume fraction of myofilaments [Vvmyo]) to a 'synthetic' (low Vvmyo) phenotype. In this altered state they respond to growth factors released from macrophages, platelets, regenerating endothelial cells and smooth muscle cells; produce large amounts of matrix; express lipoprotein scavenger receptors; express adhesion molecules for leucocytes; and express HLA-DR following exposure to the T-lymphocyte product, IFN-delta, suggesting that they can become involved in a generalised immune reaction.

Animals↗

PubMed2000

All-trans-retinoic acid limits restenosis after balloon angioplasty in the focally atherosclerotic rabbit : a favorable effect on vessel remodeling.

All-trans-retinoic acid (atRA) has potent in vitro effects on a number of processes involved in vascular injury and repair, such as modulating smooth muscle cell (SMC) proliferation and inducing SMC differentiation, and may play an important role in the in vivo response to vascular injury. We hypothesized that atRA would limit restenosis after balloon angioplasty through SMC-modulated changes in plaque size and vessel geometry. Balloon angioplasty was performed on rabbits with focal femoral atherosclerosis randomized to treatment with atRA or saline. At 28 days after balloon angioplasty, minimal luminal diameter was significantly larger in the atRA group (1.24+/-0.17 versus 1.12+/-0.22 mm, P=0.02). Histomorphometry confirmed a larger lumen area (0.51+/-0.20 versus 0. 34+/-0.13 mm(2), P=0.004) in the atRA group, with no difference in absolute plaque area. Internal elastic lamina and external elastic lamina areas were significantly larger in the atRA group (0.89+/-0. 27 versus 0.66+/-0.24 mm(2), P=0.001, and 1.29+/-0.38 versus 0. 98+/-0.32 mm(2), P=0.001, respectively). Vessel sections exhibited significantly more alpha-actin and desmin immunostaining (P=0.01) in the atRA-treated group. No differences in early cellular proliferation and collagen content were detected with the use of bromodeoxyuridine. In this atherosclerotic model of vascular injury, atRA limits restenosis after balloon angioplasty by effects secondary to overall vessel segment enlargement at the angioplasty site rather than by effects on plaque size or cellular proliferation. Increased alpha-actin and desmin immunostaining suggest a possible role for phenotypic modulation of SMCs in this favorable remodeling effect.

Actins↗

PubMed2001

Selective expression of an endogenous inhibitor of FAK regulates proliferation and migration of vascular smooth muscle cells.

Extracellular matrix signaling via integrin receptors is important for smooth muscle cell (SMC) differentiation during vasculogenesis and for phenotypic modulation of SMCs during atherosclerosis. We previously reported that the noncatalytic carboxyl-terminal protein binding domain of focal adhesion kinase (FAK) is expressed as a separate protein termed FAK-related nonkinase (FRNK) and that ectopic expression of FRNK can attenuate FAK activity and integrin-dependent signaling (A. Richardson and J. T. Parsons, Nature 380:538-540, 1996). Herein we report that in contrast to FAK, which is expressed ubiquitously, FRNK is expressed selectively in SMCs, with particularly high levels observed in conduit blood vessels. FRNK expression was low during embryonic development, was significantly upregulated in the postnatal period, and returned to low but detectable levels in adult tissues. FRNK expression was also dramatically upregulated following balloon-induced carotid artery injury. In cultured rat aortic smooth muscle cells, overexpression of FRNK attenuated platelet-derived growth factor (PDGF)-BB-induced migration and also dramatically inhibited [(3)H]thymidine incorporation upon stimulation with PDGF-BB or 10% serum. These effects were concomitant with a reduction in SMC proliferation. Taken together, these data indicate that FRNK acts as an endogenous inhibitor of FAK signaling in SMCs. Furthermore, increased FRNK expression following vascular injury or during development may alter the SMC phenotype by negatively regulating proliferative and migratory signals.

Adenoviridae↗

PubMed2005

Phenotypic heterogeneity influences the behavior of rat aortic smooth muscle cells in collagen lattice.

Phenotypic modulation of vascular smooth muscle cells (SMCs) in atherosclerosis and restenosis involves responses to the surrounding microenvironment. SMCs obtained by enzymatic digestion from tunica media of newborn, young adult (YA) and old rats and from the thickened intima (TI) and underlying media of young adult rat aortas 15 days after ballooning were entrapped in floating populated collagen lattice (PCL). TI-SMCs elongated but were poor at PCL contraction and remodeling and expressed less alpha2 integrin compared to other SMCs that appeared more dendritic. During early phases of PCL contraction, SMCs showed a marked decrease in the expression of alpha-smooth muscle actin and myosin. SMCs other than TI-SMCs required 7 days to re-express alpha-smooth muscle actin and myosin. Only TI-SMCs in PCL were able to divide in 48 h, with a greater proportion in S and G2-M cell cycle phases compared to other SMCs. Anti-alpha2 integrin antibody markedly inhibited contraction but not proliferation in YA-SMC-PLCs; anti-alpha1 and anti-alpha2 integrin antibodies induced a similar slight inhibition in TI-SMC-PCLs. Finally, TI-SMCs rapidly migrated from PCL on plastic reacquiring their epithelioid phenotype. Heterogeneity in proliferation and cytoskeleton as well the capacity to remodel the extracellular matrix are maintained, when SMCs are suspended in PCLs.

Actins↗

PubMed2002

N-cadherin upregulation and function in response of smooth muscle cells to arterial injury.

OBJECTIVE: Smooth muscle cell migration is critical to neointimal formation after arterial injury. The purpose of this study was to elucidate the regulation and functional significance of cell-cell adhesion via adherens junctions during this process. METHODS AND RESULTS: Using balloon catheter injury of rat carotid artery, we showed that neointimal formation is accompanied by dramatic but transient upregulation of intimal N-cadherin and associated catenins, proteins that mediate adhesion at adherens junctions. Upregulation was demonstrated by immunofluorescence microscopy and by immunoblotting, and it coincided with evidence of phenotypic modulation of smooth muscle cells. Similar upregulation was observed when postconfluent cultures of porcine aortic smooth muscle cells were subjected to linear denuding injuries. Furthermore, treatment of wounded cultures with a blocking antibody against the extracellular domain of the N-cadherin protein significantly suppressed the repair of wounds. CONCLUSIONS: N-cadherin and associated proteins are dynamically regulated during neointimal formation and provide evidence that this regulation is important for migratory repair. Therefore, N-cadherin may provide a novel target for therapies that are directed toward intimal proliferative disorders, including restenosis and vascular bypass graft failure.

Adherens Junctions↗

PubMed1996

Expression of a nonmuscle myosin heavy chain in glomerular cells differentiates various types of glomerular disease in rats.

To characterize the phenotypic modulation of mesangial and glomerular epithelial cells, we investigated the expression of a nonmuscle type myosin heavy chain, SMemb, and alpha-smooth muscle actin (alpha-SM actin) in rat experimental glomerular diseases, which included anti-Thy 1 nephritis, 5/6 nephrectomy, diabetes, and anti-glomerular basement membrane nephritis. SMemb was only slightly expressed in normal glomerular epithelial cells but not in mesangial cells. In the anti-Thy 1 nephritis rats, both SMemb and alpha-SM actin were most conspicuously induced in mesangial cells. However, the expression profile was shifted from alpha-SM actin to SMemb dominant pattern over the course of glomerulonephritis. The expression of SMemb was also increased in epithelial cells in this model. In the other three models, glomerular cells did not express alpha-SM actin, but did so for SMemb. In the nephrectomized and the diabetic rats SMemb was newly expressed in mesangial cells at earlier stages, but at later stages was remarkably enhanced in epithelial cells when severe glomerular hypertrophy developed. In the anti-GBM nephritis rats, SMemb expression was increased in epithelial cells. In all models examined, mesangial and epithelial expression of SMemb was confirmed by immunoelectron microscopy, and enhanced expression of SMemb mRNA in glomeruli was verified by RNase protection assay. We conclude from these results that glomerular cells change their phenotypes differently depending on various types of glomerular diseases. These phenotypic changes in glomerular cells can be revealed by the combined immunostaining for SMemb and alpha-SM actin. SMemb is especially useful to detect both mesangial and glomerular epithelial cell activation in these glomerular disease models. Understanding the functional difference and regulatory mechanisms of these cytoskeletal proteins will provide insight into the pathogenesis and progression of glomerular diseases.

Actins↗

PubMed2003

Inhibition of endothelial cell activation by the homeobox gene Gax.

BACKGROUND: cardiovascular system, strongly inhibits growth factor-stimulated phenotypic modulation of vascular smooth muscle cells in vitro and in vivo. The function of Gax in vascular endothelium is unknown, but we hypothesized that it may play a similar role there. We therefore studied Gax expression in vascular endothelial cells and its effects on proliferation and tube formation. MATERIALS AND METHODS: Gax expression in normal endothelial cells was examined in vitro by Northern blot and reverse transcriptase polymerase chain reaction and in vivo by immunohistochemistry. A replication-deficient adenovirus was then used to express Gax in human umbilical vein endothelial cells (HUVECs). HUVEC proliferation, 3H-thymidine uptake, p21 expression, and tube formation on reconstituted basement membrane were measured at different viral multiplicities of infection. RESULTS: Gax mRNA was detected in HUVECs by reverse transcriptase polymerase chain reaction and Northern blot analysis and in normal vascular endothelium by immunohistochemistry. Compared with controls transduced with a virus expressing beta-galactosidase, Gax strongly inhibited HUVEC proliferation and mitogen-stimulated 3H-thymidine uptake. p21 expression in HUVECs transduced with Gax was increased up to 5-fold as measured by Northern blot, and p21 promoter activity was activated by 4- to 5-fold. Tube formation on Matrigel was strongly inhibited by Gax expression. CONCLUSIONS: Gax is expressed in vascular endothelium and strongly inhibits endothelial cell activation in response to growth factors and tube formation in vitro. These observations suggest that Gax inhibits endothelial cell transition to the angiogenic phenotype in response to proangiogenic growth factors and, as a negative regulator of angiogenesis, may represent a target for the antiangiogenic therapy of cancer.

Blotting, Northern↗

PubMed1992

[The fibroblast, definition of its phenotype by its "programme" of biosynthesis of the extracellular matrix].

This presentation is an introduction to a symposium focusing on the fibroblast, a virtually ubiquitous cell which is cultured in many laboratories. The traditional definition of the fibroblast based solely on morphological criteria, which has satisfied most biologists for years, now needs reappraisal. The papers delivered at this symposium addressed this issue. Investigations ranged from the phylogenetic origin of fibroblasts in sponges, studied by R. Garrone, to the phenotypic modulations leading to the "myofibroblast" reported by G. Gabbiani, whose manuscript was unfortunately received too late for the conference. The present article describes how the fibroblast can be defined on the basis of its programme for producing extracellular matrix macromolecules. This programme includes a qualitative aspect, i.e., the selection of genes to be expressed: collagen types, variations in alternative splicing patterns of matrix proteins--and a quantitative aspect, i.e., the precise spatial and temporal regulation of production of "selected" macromolecules in order to meet the body's needs. This programme can be studied experimentally, and data are accumulating on the mechanisms involved and on its regulation through intrinsic factors (acting on the genome) and extrinsic and epigenetic factors (vitamins, hormones, nutrition...). Several French laboratories have supplied useful contributions to this research. The results of their investigations, which cover part of this field, are reproduced in this issue of this revue.

Collagen↗

PubMed2000

Cardiac alpha-actin in smooth muscle cells: detection in umbilical cord vessels and in atherosclerotic lesions.

Phenotypic modulation of smooth muscle cells (SMC) is a key event during the development of atherosclerotic and restenotic lesions. During this process, the composition of the cytoskeleton is substantially altered, with changes predominantly in actin expression reflecting a shift from smooth muscle alpha-actin to the non-muscle beta-isoform. We now demonstrate that yet another actin isoform, cardiac alpha-actin, is synthesized, de novo, in SMC of various atherosclerotic lesions. Using a highly specific monoclonal antibody against cardiac alpha-actin, we analyzed and compared the accumulation of this actin isoform in diverse SMC by immunofluorescence microscopy and immunoblotting. As expected, cardiac alpha-actin was present in human myocardium but not in healthy SMC of adult aorta, coronary arteries, trabeculae of the spleen, colon, stomach or skeletal muscle. Interestingly, the presence of cardiac alpha-actin was detected in umbilical cord vessels, human myometrium, in atherosclerotic coronary lesions and atherosclerotic lesions from peripheral vascular disease. The distribution of cardiac alpha-actin often paralleled that of cytokeratins 8 and 18, intermediate filament proteins typically found in dedifferentiated SMC. Taken together, the data presented here illustrate the expression of cardiac alpha-actin to be limited to either fetal vessels or those vessels or tissue having suffered damage or atrophy, outside its 'native' environment in the heart. The demonstration of cardiac alpha-actin in SMC of umbilical cord vessels and in atherosclerotic lesions but not in apparently healthy vessels supports the notion that SMC in atherosclerotic lesions exhibit a dedifferentiated phenotype.

Actins↗

PubMed1991

cAMP negatively regulates mRNA levels of actin and tropomyosin in rat cultured vascular smooth muscle cells.

The isoform expression patterns of actin and actin-binding proteins have been reported to be good markers for phenotypic modulation of rat vascular smooth muscle cells. In order to elucidate the regulatory mechanism of actin and tropomyosin isoform expression on a molecular basis, we examined the effects of various agents on the isoform expression patterns of actin and tropomyosin at the mRNA level in smooth muscle cells. We found that cAMP-elevating agents induced drastic decreases in the amounts of alpha-smooth muscle type actin and alpha-tropomyosin transcripts, the expression of other actin and tropomyosin isoforms being also repressed, but to a lesser extent. The results of the experiment involving RNA synthesis inhibitors strongly suggest that activation of some mRNA-specific degradation machinery by cAMP might be responsible at least for the rapid disappearance of alpha-smooth muscle type actin and alpha-tropomyosin transcripts in smooth muscle cells.

1-Methyl-3-isobutylxanthine↗

PubMed2001

Monocyte/mesangial cell interactions in high-glucose co-cultures.

BACKGROUND: Monocytes bind to human mesangial cells (HMC) in a co-culture model of leukocyte/ glomerular cell interactions. Since monocytic infiltration has been demonstrated in the early stages of diabetic glomerulopathy, we examined whether co-culture with myelomonocytes of the U937 cell line in media mimicking the diabetic microenvironment modulated phenotype, growth, and extracellular matrix production patterns of HMC. METHODS: HMC monolayers grown for 5 days in 5.5 mmol/l (NG) or 30 mmol/l (HG) glucose media were examined 3, 24 and 48 h after addition of U937 cells by computer-assisted image analysis/fluorescence microscopy following fixation, staining for cell adhesion, and TUNEL/propidium iodide labelling for apoptosis. As matrix components may be relevant to both phenotype of cultured HMC and monocyte adhesion, reverse transcription-polymerase chain reaction, zymography, and ELISA were used to detect urokinase-plasminogen activator (uPa), collagen type IV (COL IV), transforming growth factor beta1 (TGF-beta1), matrix metalloproteinases (MMP), and relative inhibitors (tissue inhibitor of MMP (TIMP)) expression in co-cultures in NG/HG. RESULTS: U937 adhesion at 1-3 h was increased in HG (from 54.9+/-6.6 to 87.1+/-5.8% U937/HMC). Control HMC proliferating in NG supplemented with 10% fetal bovine serum had an average cross-sectional area of 9993+/-505 micro(2) with 1.2+/-0.1 hillocks/high-power field, which increased to 13 651+/- 1114 micro(2) with 0.5+/-0.2 hillocks/high-power field in HG (P<0.05). TUNEL+HMC were nearly identical (4.9+/-1.7 vs 4.2+/-0.4% in HG, P=NS). Enhanced transcription and secretion of urokinase (uPA, +656%), COL IV (+137%), TGF-beta1 (+590%) were observed in co-cultures in HG. COL IV and TGF-beta1, but not uPA, were also increased in HMC alone, exposed to HG for 5 days. MMP-2/TIMP-2 ratio was decreased while MMP-1/TIMP-1 was increased in HG co-cultures. In both NG and HG, U937 adhesion reduced HMC number and hillocks at 24 h, with constant apoptosis. The effects of U937 were no longer detectable at 48 h, when apoptosis was 2.1+/-0.6 vs 4.0+/-0.4% in HG, and cell counts returned above basal, possibly due to a delayed proliferative response. CONCLUSIONS: High glucose medium increases U937 cell adhesion to HMC. In turn, monocytes modulate number and spatial distribution of HMC, which are also markedly affected by ambient glucose levels. These interactions may be relevant to leukocyte infiltration, mesangial expansion, and glomerulosclerosis in diabetes.

Cell Adhesion↗

PubMed2006

Benidipine, a calcium channel blocker, regulates proliferation and phenotype of vascular smooth muscle cells.

Hyperproliferation of phenotypically modified vascular smooth muscle cells (VSMCs) is one of the major factors in the development of atherosclerosis and restenosis. Previously it was demonstrated that benidipine, a dihydropyridine-calcium channel antagonist, reduced neointimal formation in a rat balloon-injury model. In the present study, we examined the effect of benidipine on the phenotypic modulation and proliferation of VSMCs, using primary cultures of rat VSMCs. In the absence of drug treatment, protein levels of the smooth muscle specific markers, such as smooth muscle myosin heavy chain-1 (SM1), calponin 1, and alpha-actin, decreased during culture. However, treatment of VSMCs with benidipine (3 - 10 micromol/L) for 1 week reversed the effect in a concentration-related manner so that high levels of marker proteins were maintained. The expression of calponin mRNAs was reduced markedly during 1-week culture, and treatment with benidipine (3 micromol/L) significantly inhibited the reduction. Treatment with benidipine for 2 days increased the level of p21 protein and partially reduced p70 S6 kinase 1 (p70S6K1) activity. These data suggest that benidipine may arrest the growth of VSMCs, thereby preventing cell dedifferentiation. These additional properties of benidipine suggest that the drug should provide useful therapy for atherosclerosis and restenosis.

Animals↗

PubMed1989

Lymphocyte proliferative response to rabies virus antigen.

Rabies virus (RV) infection, as well as active immunisation using viral antigen, elicit both humoral and cellular reactions whose protective effects are still unclear. We evaluated both responses in order to find valuable monitoring parameters for the immunisation procedure. Three laboratory workers repetitively immunised with booster human diploid cell vaccine against rabies virus, 13 patients from the anti-rabies centre (vaccinated for the first time) and 10 healthy volunteers (not immunised nor exposed to rabies virus antigen), were monitored for: (i) in vivo RV-specific antibody production; (ii) in vitro anti-RV lymphocyte proliferative response and (iii) in vitro phenotype modulation induced by the viral antigen. In particular CD3, CD4, CD8, and surface immunoglobulins were monitored. All 3 subjects receiving the booster immunisation and, to a lesser extent, those receiving 4 doses of vaccine did recognise the antigen in vitro. The proliferation involved mainly CD4 positive cells leading to an increased number of cell bearing surface immunoglobulins, i.e. B cells. The proliferation index was in good correlation with the in vivo antibody production (p = less than 0.00009441). Nevertheless the presence of some cases without correlation between those parameters (in particular 5 out of 6 patients over 65 years of age failed to mount an adequate cellular proliferative response) reveals the need to use cellular response in parallel to the current humoral response, in order to evaluate and monitor the immunisation procedure. This conclusion is further stressed by the fact that protection against rabies infection is mainly cellular.

Adolescent↗

PubMed2006

Regulation of vascular smooth muscle proliferation and migration by beta2-chimaerin, a non-protein kinase C phorbol ester receptor.

The proliferation and migration of vascular smooth muscle cells (SMC) are important aspects of atherogenesis. Activated growth factor signaling in injured vessels subsequently promotes a number of intracellular events resulting in the phenotypic modulation of SMC. Here, we investigated the role of beta2-chimaerin, a non-protein kinase C phorbol ester receptor with Rac-GTPase-activating protein activity, in growth factor-stimulated SMC. The endogenous expression of beta2-chimaerin was detected in cultured human SMC by reverse transcription-polymerase chain reaction and immunohistochemistry. Next, the overexpression of HA-tagged wild-type human beta2-chimaerin was attempted using cultured rat SMC with a recombinant adenovirus (Adv-beta2-Chim). Adv-LZ encoding beta-galactosidase (LacZ) was used as the control. The proliferation of SMC stimulated by platelet-derived growth factor (PDGF-BB, 10 ng/ml), as measured by cell-counting and 5-bromo-2'deoxyuridine incorporation assay, was suppressed by infection with Adv-beta2-Chim (50-200 MOI), but not with control viruses. PDGF-induced SMC migration was inhibited by approximately 25% after infection with Adv-beta2-Chim (200 MOI) using a modified Boyden's chamber assay with a fibronectin-coated membrane. Confocal microscopy revealed that PDGF stimulation altered the sub-cellular localization of beta2-chimaerin. The administration of 12-O-tetradecanoyl phorbol 13-acetate also induced changes in the sub-cellular localization of beta2-chimaerin, which was not affected by a presence of the PKC inhibitor (GF109203X). Finally, PDGF-induced Rac1 activation was found to be inhibited in the Adv-beta2-Chim-infected cells. Thus, we demonstrated that beta2-chimaerin regulates the proliferation and migration of SMC downstream of growth factor signaling pathway via the regulation of Rac1 activity. The signaling mediated by beta2-chimaerin may play a role in the regulation of SMC phenotypes, thereby implicating human atherogenesis.

Animals↗

PubMed2004

Estrogenic restoration of functional pancreatic islet cytoarchitecture in diabetes (db/db) mutant C57BL/KsJ mice: relationship to estradiol localization, systemic glycemia, and persistent hyperinsulinemia.

The diabetes (db/db) genotype mutation induces a hyperglycemic-hyperinsulinemic endometabolic state in C57BL/KsJ mice, manifesting a type 2 NIDDM diabetes-obesity syndrome (DOS) in this hyperphagic, leptin receptor (lf) defective model. The severity of the DOS induced by the single gene, homozygous-recessive mutation may be therapeutically moderated by gonadal steroids and pre-steroidal metabolites. The current studies define the estradiol (E2)-modulated phenotypic, systemic, cytochemical, and cellular metabolic responses to db/db mutation expression as compared to littermate control (+/?) indices. The db/db mutation induced dramatic age- and DOS severity-related increases in body weights, blood glucose, and serum insulin concentrations relative to +/? indices between 4-week-old (i.e., initial onset stage of DOS phenotype) and 16-week-old (i.e., chronic stage of DOS) groups. Chronic, low-dose (0.1 microg/3.5 days) E2 treatment (E2-HRx) significantly reduced the obesity mass and blood glucose levels of db/db mutants relative to oil-HRx groups. Similarly, E2-HRx maintained pancreatic glucose utilization rates and pancreatic tissue weights in db/db mutants to near +/? indices. Concurrent amelioration of db/db-enhanced pancreatic lipogenesis and islet hypercytolipidemia occurred following E2-HRx. Pancreatic islet lipo-deposition was markedly reduced in db/db mutants following E2-HRx, and the restoration of islet size and cellular insulin concentrations correlated with beta-cell cytoplasmic regranulation of insulin secretory vesicles. In chronic E2-HRx db/db groups, autoradiographic localization of (3)H-E2 was demonstrated in the nuclear compartments of regranulated, nonhypertrophic islet cell populations, including insulin-containing beta-cells. In chronic E2-HRx db/db mutants, beta-cell insulin granulation was prominent in mildly hypertrophic pancreatic islets, with cytodistribution patterns and concentrations comparable to normal +/? indices. In contrast, E2-HRx maintained the systemic hyperinsulinemia characteristic of oil-HRx db/db mutants. The results of these studies indicate that the severity of the type 2 NIDDM endometabolic syndrome induced by the db/db genotypic mutation may be influenced by E2-HRx, including reduction of the islet hypercytolipidemia and hypertrophic atrophy which are indicators of impending pancreatic involution in this mutant model. The hypercytolipidemia-induced demise of beta-cell cytoarchitecture was reduced by E2-HRx, including the reestablishment of islet beta-cell cytogranulation. These data suggest that the severity of genomic db/db-mutation expression may be modified by E2-HRx, with the gonadal steroid probably acting as a nuclear-specific stimulatory transcriptional modulator of cellular glucometabolic cascades in the absence of leptin-directed homeostatic influences.

Acetyl-CoA Carboxylase↗