Coronary stent implantation changes 3-D vessel geometry and 3-D shear stress distribution

Mechanisms of in-stent restenosis are not fully understood. Shear stress is known to play a role in plaque and thrombus formation and is sensitive to changes in regional vessel geometry. Hence, we evaluated the regional changes in 3-D geometry and shear stress induced by stent placement in coronary arteries of pigs.Methods. 3-D reconstruction was performed, applying a combined angiographic and IVUS technique (ANGUS), from seven Wallstents (diameter 3.5 (n=3) and 5mm (n=4)), which were implanted in seven coronary arteries of five pigs. This 3-D geometry was used to calculate locally the curvature, while the shear stress distribution was obtained by computational fluid dynamics. Local changes in shear stress were obtained at the entrance and exit of the stent for baseline (0. 65+/-0.22 ml/s) and hyperemic flow (2.60+/-0.86 ml/s) conditions. Results. After stent implantation, the curvature increased by 121% at the entrance and by 100% at the exit of the stent, resulting in local changes in shear stress. In general, at the entrance of the stent local maxima in shear stress were generated, while at the exit both local maxima and minima in shear stress were observed (p<0.05). Additionally, the shear stress at the entrance and exit of the stent were correlated with the local curvature (r: 0.30-0.84).Conclusion. Stent implantation changes 3-D vessel geometry in such a way that regions with decreased and increased shear stress occur close to the stent edges. These changes might be related to the asymmetric patterns of in-stent restenosis.     

Coordinate regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and prenyltransferase synthesis but not degradation in HepG2 cells

Human hepatoma HepG2 cells were used to demonstrate coordinate regulation of three enzymes of cholesterol synthesis under a variety of conditions. Addition of either delipidized serum and mevinolin or low density lipoprotein, 25-hydroxycholesterol, or mevalonic acid to HepG2 cells resulted in rapid changes both in the levels of the mRNAs and in the rates of synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, HMG-CoA reductase, and farnesyl pyrophosphate synthetase (prenyltranferase). In all cases, the changes in mRNA levels were paralleled by changes in the rates of specific protein synthesis. Pulse-chase techniques were used to determine the half-lives of all three proteins. Addition of low density lipoprotein to the media during the chase increased the rate of degradation of HMG-CoA reductase 4.6-fold but had no affect on the half-lives of HMG-CoA synthase or prenyltransferase. Therefore, we conclude that the coordinate regulation of these three enzymes under a variety of conditions occurs at the level of enzyme synthesis and not at the level of protein stability.     

Interaction of 3-hydroxybenzoate with 3-hydroxybenzoate-6-hydroxylase.

The gradual quenching of the emission fluorescence of 3-HBA in the visible region upon titration with 3-HBA-6-hydroxylase and distinct changes in the near-UV circular dichroic spectrum of the enzyme in the presence of substrate suggest the formation of a stable enzyme-substrate complex. The binding of aromatic substrate 3-hydroxybenzoate to 3-hydroxybenzoate-6-hydroxylase occurs without gross changes in the backbone structure of the enzyme. The binding strength of the ES complex is partially reduced upon chemical modification of arginine, histidine, or tryptophan residues of enzyme, probably implicating their concerted action in the binding of substrate to enzyme. Partial inactivation of enzyme and diminished stability of the ES complex in response to treatment with 1 M urea could be ascribed to localized effects of the denaturant.     

Induction of succinyl-coenzyme A:3-oxoacid coenzyme A-transferase during differentiation of 3T3-L1 cells

Differentiation of confluent 3T3-L1 preadipocytes to adipocytes in the presence of dexamethasone and 1-methyl-3-isobutylxanthine for 7 days resulted in a 4-fold increase in the incorporation of acetoacetate-carbon into fatty acids and in the activity of 3-oxoacid CoA-transferase, which catalyzes the first committed step in the conversion of acetoacetate to acetoacetyl-CoA. The increase in enzyme activity was due to an increase in the cellular content of the enzyme, as determined by immunoprecipitation of 3-oxoacid CoA-transferase from 3T3-L1 preadipocytes and adipocytes with rabbit antiserum specific for the rat brain enzyme. The 4-fold increase in enzyme activity was accompanied by a 2.7-fold increase in the average relative rate of synthesis of 3-oxoacid CoA-transferase (between Days 4 and 7). Additionally, the half-life of the enzyme increased 1.9-fold relative to the half-life of total protein, indicating that changes in both synthesis and degradation of 3-oxoacid CoA-transferase are responsible for alterations in its activity. Previous studies on the turnover of other enzymes that are induced during differentiation of 3T3-L1 cells have assigned changes in enzyme synthesis as the primary or sole mechanism for changes in enzyme activity. This report provides the first documentation that both enzyme synthesis and degradation play a role in regulating the enzyme activity of an enzyme during differentiation of 3T3-L1 cells.     

Phenotypic changes in 3T3 cells associated with the change of sphingolipid synthesis by a ceramide analog, 2-decanoylamino-3-morpholino-1-phenylpropanol (compound RV538)

A culture of BALB/c 3T3 cells grown in the presence of 40 microM of the ceramide analog compound RV538 (2-decanolyamino-3-morpholino-1-phenylpropanol) for several passages caused a substantial decrease in the level of all glycosphingolipids and an accumulation of ceramide and sphingomyelin. Associated with these chemical changes of sphingolipid composition and metabolism, the following phenotypic changes were observed: (i) loss of the cobblestone appearance at cell density saturation and development of fibroblastic appearance with partial overlapping of cells; (ii) reduction of cell growth rate; (iii) enhanced production of lactic acid; (iv) enhanced rate of glucose transport; and (v) higher incidence of large colony formation with infiltrating appearance in soft agar. Cell morphology changes, lactate production, and enhanced sugar uptake were reversed by co-culturing cells with gangliosides, particularly trisialogangliosides. Thus, these phenotypic changes mimicking those of oncogenically transformed cells are closely related to the blocked synthesis of glycolipids in these cells, whereas other changes may be caused by an accumulation of ceramide and sphingomyelin.     

Analysis of gene expression profile during 3T3-L1 preadipocyte differentiation

Cellular differentiation is a process in which a group of differentiation specific genes is programmatically induced. This gene expression program leads to changes in both cellular morphological and physiological phenotypes. Using an 18,376-member cDNA/EST microarray, we analyzed the difference in gene expression profiles between differentiated 3T3-L1 adipocyte and non-differentiated 3T3-L1 preadipocyte. From our study a large number of genes and ESTs were identified as differentially induced or suppressed. In this paper we describe the changes of gene expression profile during 3T3-L1 cell differentiation.     

Isolation of Swiss 3T3 cell variants with altered heparan sulfate

The replica filter technique has been used to isolate variants of Swiss mouse 3T3 cells which produce heparan sulfates with altered levels of sulfation. These changes in the extent of sulfation correlate with alterations in cell morphology, in the organization of cytoskeletal elements, focal contacts, and the extracellular matrix, and in the growth regulation of cells, as expressed by saturation density. An increase in the extent of heparan sulfate sulfation occurs concomitantly with a decreased saturation density and enhanced focal contact formation. In contrast, graded decreases in sulfation correlate with graded increases in saturation density and losses of cytoskeletal and extracellular matrix organization. These graded responses appear very similar to those which have been reported for the transformation of cells with fusiform mutants of Rous sarcoma virus or the adenovirus type 2 Ela transforming gene and suggest that the morphological changes observed in the transformed cells can be controlled by cellular systems.     

New indices in morphometry of nuclear structure in the NIH 3T3 cell line

We analyzed the connection of changes in nucleus ploidy with changes in nucleolar apparatus of NIH 3T3 cells. The quantity of nucleoli does not depend on the quantity of nucleolar DNA, but instead depends on euploidy: the majority of euploid cells have 1-3 nucleoli. The quantity of DNA in the nucleolus is correlated with the quantity of nucleolar DNA, and does not depend on ploidy changes. The nucleolar area has a tendency to increase in line with an increase in their numbers in the nucleus. The relationship of the quantity of DNA in the nucleolus with that of the nucleus is stable. During the process of increase in the number of nucleoli in a nucleus, there is a corresponding decrease in the quantity of DNA in each nucleolus, and there is likewise no increase in the sum of nucleolar DNA. The ratio of sums of the nucleolar perimeters to nuclear perimeter is a significant factor, which increases linearly along with an increase in the number of nucleoli in a nucleus.     

Aggregation of poly(γ-benzyl-L-glutamate) in mixed solvent systems

The aggregation of poly(γ-benzyl L -glutamate) has been studied by measuring the specific Kerr constants (B/c) over a tenfold range of concentration and the intrinsic viscosities of solutions of a low molecular weight sample of the polymer in four solvent mixtures, viz., benzene–dimethylformamide (DMF), benzene–ethylene dichloride (EDC), dioxane–DMF and dixoane–EDC. Sharp changes are found in the experimentally determined quantities on the addition of small amounts of polar solvent to solutions of the polymer in either benzene or dioxane; this implies that lyotropic phase changes are occuring. The aggregation in benzene produces a birefringent, viscous solution which is probably a smeetic liquid crystal. This changes on addition of polar solvent to an aggregation involving only a few molecules; the second aggregate is most likely antiparallel. Aggregation in dioxane is antiparallel; the existence of a smectic phase is ruled out by the low intrinsic viscosities.     

Lipid composition of Balb/c3T3, SV3T3, and Concanavalin A-selected revertant cells grown in media containing lipid-depleted serum

The effects of growth in media supplemented with lipid-depleted fetal calf serum (LDS-media) on morphology, saturation density, and lipid composition were studied in Balb/c3T3, SV3T3, and Concanavalin A selected SV3T3 revertant cells (SV3T3 Rev cells). Cells grown in media containing complete fetal calf serum (FCS-medium) or reconstituted FCS (RS-medium) were used as controls. Growth in LDS-media reduced saturation densities of both SV3T3 and SV3T3 Rev cells while it affected only slightly the saturation density of normal parental cells. Similar inhibitory effects on growth were also induced by exposure of RS-medium. Growth in LDS-medium did not change the typical morphology of the three cell lines. 3T3, SV3T3, and SV3T3 Rev cells grown in LDS-medium showed an accumulation of triacylglycerols and free fatty acids together with a reduction of free cholesterol. All these changes were also present, however, in cells grown in these changes were also present, however, in cells grown in RS-Medium. Growth in LDS-medium induced an increase of 16:1 and 18:1, a decrease of 20:4, and an accumulation of 20:3 (n-9) in phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol + phosphatidylserine of 3T3 cells. By contrast, only a slight accumulation of 20:3 (n-9) accompanied by a moderate increase of monoenoic acids was found in the phospholipids of SV3T3 cells grown in LDS-medium. SV3T3 Rev cells grown in LDS-medium showed changes in phospholipid fatty acids composition similar to those found in SV3T3 cells grown under the same conditions.     

Helix–coil transition in copolypeptides. II. Poly(γ-benzyl L-glutamate-co-ε-carbobenzoxy-L-lysine)

The thermal helix–coil transition of poly(γ-benzyl L -glutamate-co-ε-carbobenzoxy-L -lysine) copolypeptides was studied in solvent mixtures of different compositions. The cooperativity parameter v changes linearly with polymer (and solvent) composition, whereas the heat of the transition shows a very pronounced minimum as a function of polymer composition. This minimum cannot be due only or mainly to the solvent changes and must be attributed to the effect on the transition of the side chains of the polypeptides.     

Uptake of putrescine by 3T3 and SV3T3 cells and its effect on ornithine decarboxylase activity

Arterial smooth muscle cells undergo marked biochemical and morphological changes upon culturing. We have studied the time course of these changes in smooth muscle cells isolated from normal rabbit aortas by enzymic digestion and then maintained in Dulbecco's modified Eagle's medium with or without 10% rabbit serum. Subcultured smooth muscle cells were also examined. Isolated cells cultured in the presence of serum multiply rapidly and by 9 days exhibit features typical of subcultured cells including multilayered growth, elevated marker enzyme activities of subcellular organelles, and proliferation of organelles. In contrast, isolated cells cultured in the absence of serum remain quiescent, as indicated by the low level (<10%) of ₃H-thymidine incorporation into nuclei and constant DNA content of the cultures. These cells spread slowly to form a monolayer of randomly oriented cells and they retain differentiated morphological features. Their enzyme activities remain at the levels of those of freshly isolated cells initially, but by 5 days some enzyme activities increase, in particular those of the acid hydrolases and catalase. Rates of pinocytosis and protein synthesis in these cells are comparable to those of cells maintained in serum-supplemented medium for the same period, but are significantly less than those measured in subcultured cells. Within 5 days, morphological alterations in the serum-deprived cells occur including the presence of increased numbers of lysosomes. Quiescent cultures of enzymically isolated cells may be a useful tool for short-term biochemical and physiological studies of differentiated arterial smooth muscle cells.     

The high rate of aerobic glycolysis of transformed 3T3 cells persists after cell homogenization.

The high rate of lactic acid production by 3T3 cells which have been transformed by simian virus 40 or by polyoma virus as compared to confluent untransformed 3T3 cells persists after cell homogenization. This difference is also reflected by increased phosphofructokinase activity in the viral transformed cells. The findings imply that the increased aerobic glycolysis in the transformed cells results from changes in the glycolytic pathway rather than changes in sugar transport.     

Gene expression changes associated with the anti-angiogenic activity of kallikrein-related peptidase 3 (KLK3) on human umbilical vein endothelial cells

Kallikrein-related peptidase 3 (KLK3, also known as prostate-specific antigen, PSA) is a chymotrypsin-like kallikrein that has anti-angiogenic properties. We have previously shown in a human umbilical vein endothelial cell (HUVEC) model that the anti-angiogenic effect of KLK3 is related to its enzyme activity. However, the mechanism of this effect remains to be clarified. To this end, we used a DNA microarray to study KLK3-induced changes in gene expression associated with reduction of HUVEC tube formation. Among the 41,000 genes studied, 311 were differentially expressed between control and KLK3-treated cells. These changes were enriched in several pathways, including those associated with proteasome, ubiquitin-mediated proteolysis, focal adhesion and regulation of the actin cytoskeleton. Furthermore, the changes were opposite to those previously described to occur during tubulogenesis. In conclusion, our results show that KLK3 induces gene expression changes in HUVECs. Although these changes might be relevant for the mechanism by which KLK3 exerts its anti-angiogenic activity, it cannot be judged from the present results whether they reflect the primary mechanism mediating the effect of KLK3 or are secondary to morphogenic differentiation.     

Effect of plasma membranes on solute transport in 3T3 cells.

Sparse cultures of Swiss 3T3 cells are arrested early in the G1 phase of growth by the addition of a plasma membrane fraction obtained from confluent 3T3 cells. We have examined whether the changes in solute transport which are usually associated with cessation of growth at confluency also take place when cell growth is arrested by the addition of plasma membranes. We find that the rate of uptake of alpha-aminoisobutyric acid and uridine is decreased after the addition of plasma membranes to 3T3 cells, but the rate of uptake of 2-deoxyglucose and phosphate is not. We conclude from these observations that uptake of uridine and alpha-aminoisobutyric acid are related to contact inhibition of growth, while the decline in the rate of uptake of 2-deoxyglucose and phosphate observed at high cell density must be due to changes other than cell to cell contact.     

14-3-3zeta C-terminal stretch changes its conformation upon ligand binding and phosphorylation at Thr232

14-3-3 proteins are abundant binding proteins involved in many biologically important processes. 14-3-3 proteins bind to other proteins in a phosphorylation-dependent manner and function as scaffold molecules modulating the activity of their binding partners. In this work, we studied the conformational changes of 14-3-3 C-terminal stretch, a region implicated in playing a role in the regulation of 14-3-3. Time-resolved fluorescence and molecular dynamics were used to investigate structural changes of the C-terminal stretch induced by phosphopeptide binding and phosphorylation at Thr232, a casein kinase I phosphorylation site located within this region. A tryptophan residue placed at position 242 was exploited as an intrinsic fluorescence probe of the C-terminal stretch dynamics. Other tryptophan residues were mutated to phenylalanine. Time-resolved fluorescence measurements revealed that phosphopeptide binding changes the conformation and increases the flexibility of 14-3-3zeta C-terminal stretch, demonstrating that this region is directly involved in ligand binding. Phosphorylation of 14-3-3zeta at Thr232 resulted in inhibition of phosphopeptide binding and suppression of 14-3-3-mediated enhancement of serotonin N-acetyltransferase activity. Time-resolved fluorescence of Trp242 also revealed that phosphorylation at Thr232 induces significant changes of the C-terminal stretch conformation. In addition, molecular dynamic simulations suggest that phosphorylation at Thr232 induces a more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. These results indicate that the conformation of the C-terminal stretch plays an important role in the regulation of 14-3-3 binding properties.