Coordination in gene expression during atherogenesis

General tendencies in the regulation of gene expression during atherogenesis were investigated using correlation analysis for 34 mRNA species of several functional groups. The contents of mRNA were measured by quantitative PCR in samples of human aortal intima containing no lesions or atherosclerotic lesions of types I (initial lesions), II (fatty streaks), and Va (fibroatheromas). The coupling between mRNA contents in lesions and the same mRNAs in intact tissue was found to descend in the course of the disease progression. The data are in accordance with the opinion that successive morphologic types of atherosclerotic lesions correspond to steps of atherogenesis. In addition, the contents of individual mRNA species could correlate with each other within the given sample type, the extent of this coupling rising along with the disease progression. The exception from this rule was a collapse in coupling for several functional groups of mRNA in lesions of type I. This collapse could indicate special position of these lesions in pathogenesis. Statistically significant correlations between mRNAs found in samples of all four types comprised in total about 50% of all possible correlations. 66% of these correlations were conservative, i.e. observed in at least two sample types. By coupling-strength, the studied mRNAs could be divided into four clusters whose composition significantly varied along with the disease progression. The disease progression was also associated with decline in number of regulatory factors that determine coordination in expression of the analyzed genes.     

The effect of calcium antagonists on cholesterol metabolism in human aortal intima cells and macrophage lines]

Effects of two Ca-antagonists, verapamil and nifedipine, on the total cellular cholesterol content and accumulation, as well as on the synthesis and hydrolysis of cholesteryl esters in human aortic intimal smooth muscle cells and P388D1 cell line have been studied. Verapamil and nifedipine used at 10(-6) M and higher concentrations decreased the total cellular cholesterol content (by 25-40%) in intimal cells isolated from atherosclerotic lesions without any effect on the cholesterol content in normal intimal cells or P388D1 cells. At 2 x 10(-5) M verapamil and nifedipine prevented the accumulation of cholesterol induced by atherogenic blood serum or atherogenic low density lipoproteins in both types of cells. At 10(-5) M and higher concentrations verapamil and nifedipine inhibited (2-3-fold) cholesteryl ester synthesis in intimal cells and, used at 10(-6) M and higher doses, in P388D1 cells as well. Verapamil and nifedipine (2 x 10(-5) M) enhanced the hydrolysis of cholesteryl esters in both types of cells. The Ca-channel agonist Bay K8644 had no effect on cholesteryl ester synthesis, nor did it suppress its inhibition by Ca-antagonist. The beta-receptor blocker propranolol induced the accumulation of cholesterol in intimal cells and inhibited the synthesis and hydrolysis of cholesterol esters in these cells. The data obtained suggest that the antiatherosclerotic action of Ca-blockers is determined by their ability to reduce the cellular cholesterol content which is suggested to be the result of enhanced hydrolysis of cellular cholesteryl esters.     

An improved microindentation technique to measure changes in properties of arterial intima during atherogenesis

A fully automated instrument was developed that measured the amount and time-course of indentation of arterial intima by a spherical tipped probe 25 micrometers in diameter loaded routinely by forces of 10 microN. The relationship of depth of indentation to time was non-exponential but reached an apparent asymptotic value in about 6 s. Routine indentations were recorded 10 s after loading, the relationship of indentation and force being essentially linear between 0-100 microN. The instrument is an order of magnitude more sensitive than one previously described (Gow and Vaishnav, 1975, J. Appl. Physiol. 38, 344-350) and produces indentations of 1-4 micrometers in the intima of rabbit thoracic aorta with a reproducibility of 1-2%. Preliminary usage of the new microindentor has shown increases in the compliance of the thoracic aorta intima in rabbits during cholesterol feeding.     

Changes in plant gene expression during stress

Changes in gene expression which occur during periods of environmentally induced stress provide models for the study of gene regulation. Several types of stress have been shown to elicit a specific and reproducible pattern of gene expression in various plant species. These stress factors include heat shock, anaerobiosis, plant pathogens, oxygen free radicals, heavy metals, water stress, and chilling. In some cases, changes in specific genes have been identified, such as increases in the expression of the gene encoding the phytoalexin-synthesizing enzyme in pathogen elicitor-treated cells. However, in most cases, the functional identity of stress-induced genes is unknown. The alterations in gene expression during stress usually are rapid and repeatable, making these genetic systems ideal for examination of factors and mechanisms involved in gene regulation.     

Changes in glycolytic enzyme levels and isozyme expression in human lymphocytes during blast transformation.

The levels of each of the glycolytic enzymes were observed to exhibit a parallel increase of 200 to 300% when human lymphocytes were stimulated to undergo blast transformation. A series of electrofocusing and electrophoretic studies was utilized to assess the isozyme distribution of the glycolytic enzymes during blastogenesis. Hexokinase (pI = 7.40), glucosephosphate isomerase (pI = 9.35), and enolase (pI = 8.30) existed as single electrophoretic components and were unchanged during blast transformation. Phosphoglycerate mutase was observed to exist as two isozymes (pI = 5.80 and 6.63), which were also unchanged by blastogenesis. Aldolase, which was present as two electrophoretic forms in lymphocytes (pI = 9.25 and 8.75), exhibited a shift in the relative content of each. In addition to the lactate dehydrogenase isozymes at pI 9.50 and 7.60 found in lymphocytes, lymphoblasts contained isozymes with pI values of 7.30, 7.05, and 5.85. Although glyceraldehyde 3-phosphate dehydrogenase was present as a single electrophoretic form (pI ? 8.0) in both lymphocytes and lymphoblasts, the association of the enzyme with actin produced electrophoretic artifacts with lower pI values. Phosphoglycerate kinase, which appeared as a single form in lymphocytes (pI = 9.00), was present as two isozymes (9.00 and 8.74) in lymphoblasts. Similarly, pyruvate kinase (pI = 8.73 and 8.50 in lymphocytes) exhibited additional isozymes (pyruvate kinase, pI = 7.60 and 5.85, and triosephosphate isomerase, pI = 5.20) as a result of cell transformation.     

Changes in gene expression during senescence of adrenocortical cells in culture

Bovine adrenocortical cells undergo a process in which expression of steroid hydroxylases is lost progressively as a function of population doubling level (PDL) in culture. Each cytochrome P450 shows a characteristic rate of loss of expression as a function of PDL (in order of rates of loss: CYP11B > CYP21 > CYP17 > CYP11A). CYP11B and CYP21 require insulin-like growth factor I as well as cyclic AMP; these are the only factors required for induction in the primary culture. Middle- and later passage cells do not express CYP11B and CYP21 under the same conditions, but will do so when cells are grown in extracellular matrix Matrigel. In late-passage cells neither CYP17, CYP21, nor CYP11B are expressed, even in the presence of Matrigel; only CYP11A is expressed in late-passage cultures. When the different environmental factors required for induction of CYP11B and CYP21 are taken into account, induction of these genes disappears with the same kinetics as previously shown for CYP17 as a function of PDL. The primary cause of the loss of expression of these genes is likely to be a phenotypic switching event similar to that previously demonstrated for CYP17 by in situ hybridization. The mechanism of phenotypic switching is unknown. However, one HpaII site at −2.3 kb of CYP17 was methylated in the bovine adrenal cortex in vivo but showed rapid and complete demethylation when adrenocortical cells were placed in culture. This indicates a unique, reproducible, environmentally determined change in methylation, with as yet undetermined consequences. However, data from reporter constructs suggest that phenotypic switching does not result from a simple loss of regulatory factors that act within 2.5 kb of the promoter. Previous data suggested that SV40 T antigen may affect phenotypic switching, and thus that SV40 may be useful for the derivation of functional adrenocortical cell lines. Adaptation of methods previously used for bovine cells to human adrenocortical cells to produce SV40 T antigen-transfected clones yielded data indicating preservation of essential aspects of the human adrenocortical cell differentiated phenotype.     

Alteration of gene expression levels during osteogenic induction of human adipose derived stem cells in long-term culture

Adipose tissue is a source of multipotent stem cells and it has the ability to differentiate into several types of cell lineages such as neuron cells, osteogenic and adipogenic cells. Most studies on human adipose-derived stem cells (ASCs) have been carried out at the early passages. For clinical usage, ASCs need to be expanded in vitro for a period of time to get sufficient cells for transplantation into patients. However, the impact of long-term culture on ASCs molecular characteristics has not been established yet. Several studies have also shown that osteogenic and adipogenic cells have the ability to switch pathways during in vitro culture as they share the same progenitor cells. This data is important to ensure their functionality and efficacy before being used clinically in the treatment of bone diseases. Therefore, we aim to investigate the effect of long-term culture on the adipogenic, stemness and osteogenic genes expression during osteogenic induction of ASCs. In this study, the molecular characteristics of ASCs during osteogenic induction in long-term culture was analysed by observing their morphological changes during induction, analysis of cell mineralization using Alizarin Red staining and gene expression changes using quantitative RT-PCR. Morphologically, cell mineralization at P20 was less compared to P5, P10 and P15. Adipogenesis was not observed as negative lipid droplets formation was recorded during induction. The quantitative PCR data showed that adipogenic genes expression e.g. LPL and AP2 decreased but PPAR-γ was increased after osteogenic induction in long-term culture. Most stemness genes decreased at P5 and P10 but showed no significant changes at P15 and P20. While most osteogenic genes increased after osteogenic induction at all passages. When compared among passages after induction, Runx showed a significant increased at P20 while BSP, OSP and ALP decreased at later passage (P15 and P20). During long-term culture, ASCs were only able to differentiate into immature osteogenic cells.     

Changes in keratin gene expression during terminal differentiation of the keratinocyte

Cells of the inner layers of the epidermis contain small keratins (46-58K), whereas the cells of the outer layers contain large keratins (63-67K) in addition to small ones. The changes in keratin composition that take place within each cell during the course of its terminal differentiation result largely from changes in synthesis. Cultured epidermal cells resemble cells of the inner layers of the epidermis in synthesizing only small keratins. The cultured cells possess translatable mRNA only for small keratins, whereas mRNA extracted from whole epidermis can be translated into both large and small keratins. As no synthesis takes place in the outermost layer of the epidermis (stratum corneum), the keratins of this layer must be synthesized earlier, but in some cases they then become smaller: this presumably occurs by post-translational processing of the molecules during the final stages of differentiation. Stratified squamous epithelia of internal organs do not form a typical stratum corneum and do not make the large keratins characteristic of epidermis. Their keratins are also different from those of cultured keratinocytes, implying that they have embarked on an alternate route of terminal keratin synthesis.     

Control of gibberellin levels and gene expression during de-etiolation in pea

Gibberellin A(1) (GA(1)) levels drop significantly in wild-type pea (Pisum sativum) plants within 4 h of exposure to red, blue, or far-red light. This response is controlled by phytochrome A (phyA) (and not phyB) and a blue light receptor. GA(8) levels are increased in response to 4 h of red light, whereas the levels of GA(19), GA(20), and GA(29) do not vary substantially. Red light appears to control GA(1) levels by down-regulating the expression of Mendel's LE (PsGA3ox1) gene that controls the conversion of GA(20) to GA(1), and by up-regulating PsGA2ox2, which codes for a GA 2-oxidase that converts GA(1) to GA(8). This occurs within 0.5 to 1 h of exposure to red light. Similar responses occur in blue light. The major GA 20-oxidase gene expressed in shoots, PsGA20ox1, does not show substantial light regulation, but does show up-regulation after 4 h of red light, probably as a result of feedback regulation. Expression of PsGA3ox1 shows a similar feedback response, whereas PsGA2ox2 shows a feed-forward response. These results add to our understanding of how light reduces shoot elongation during de-etiolation.     

Changes in Leydig cell gene expression during development in the mouse

Developmental changes in the expression of 18 Leydig cell-specific mRNA species were measured by real-time polymerase chain reaction to partially characterize the developmental phenotype of the cells in the mouse and to identify markers of adult Leydig cell differentiation. Testicular interstitial webs were isolated from mice between birth and adulthood. Five developmental patterns of gene expression were observed. Group 1 contained mRNA species encoding P450 side chain cleavage (P450(scc)), P450(c17), relaxin-like factor (RLF), glutathione S-transferase 5-5 (GST5-5), StAR protein, LH receptor, and epoxide hydrolase (EH); group 2 contained 3beta-hydroxysteroid dehydrogenase (3beta-HSD) VI, 17beta-hydroxysteroid dehydrogenase (17beta-HSD) III, vascular cell adhesion molecule 1, estrogen sulfotransferase, and prostaglandin D (PGD)-synthetase; group 3 contained patched and thrombospondin 2 (TSP2); group 4 contained 5alpha-reductase 1 and 3alpha-hydroxysteroid dehydrogenase; group 5 contained sulfonylurea receptor 2 and 3beta-HSD I. Group 1 contained genes that were expressed in fetal and adult Leydig cells and which increased in expression around puberty toward a maximum in the adult. Group 2 contained genes expressed only in the adult Leydig cell population. Group 3 contained genes with predominant fetal/neonatal expression in the interstitial tissue. Group 4 contained genes with a peak of expression around puberty, whereas genes in group 5 show little developmental change in expression. Highest mRNA levels in descending order were RLF, P450(c17), EH, 17beta-HSD III, PGD-synthetase, GST5-5, and P450(scc). Results identify five genes expressed in the mouse adult Leydig cell population, but not in the fetal population, and one gene (TSP2) that may be expressed only in the fetal Leydig cell population. The developmental pattern of gene expression suggests that three distinct phases of adult Leydig cell differentiation occur.     

Changes in free polyamines and gene expression during peach flower development

Free polyamine contents and expressions of five genes encoding for polyamine biosynthetic enzymes were investigated at four different stages during peach (Prunus persica L. Batsch cv. Akatsuki) flower development. Fresh mass of peach flowers increased, accompanied by reduction in contents of total polyamines and putrescine/spermidine ratio due to decrease in putrescine content. Spermidine, the largest fraction, and spermine, the least part, underwent minor change. Expressions of the five key genes involved in polyamine biosynthesis during flower development did not parallel the changes in free polyamines.     

Plasma levels and cardiovascular gene expression of urotensin-II in human heart failure

The peptide urotensin-II (U-II) has been described as most potent vasoconstrictor identified so far, but plasma values in humans and its role in cardiovascular pathophysiology are unknown. We investigated circulating urotensin-II and its potential role in human congestive heart failure (CHF). We enrolled control individuals (n=13; cardiac index [CI], 3.5+/-0.1 l/min/m2; pulmonary wedge pressure [PCWP], 10+/-1 mm Hg), patients with moderate (n=10; CI, 2.9+/-0.3 l/min/m2; PCWP, 14+/-2 mm Hg) and severe CHF (n=11; CI, 1.8+/-0.2 l/min/m2; PCWP, 33+/-2 mm Hg). Plasma levels of urotensin-II differed neither between controls, patients with moderate and severe CHF nor between different sites of measurement (pulmonary artery, left ventricle, coronary sinus, antecubital vein) within the single groups. Hemodynamic improvement by vasodilator therapy in severe CHF (CI, +78+/-3%; PCWP, -55+/-3%) did not affect circulating U-II over 24 h. Preprourotensin-II mRNA expression in right atria, left ventricles, mammary arteries and saphenous veins did not differ between controls with normal heart function and patients with end-stage CHF. In conclusion, urotensin-II plasma levels and its myocardial and vascular gene expression are unchanged in human CHF. Circulating urotensin-II does not respond to acute hemodynamic improvement. These findings suggest that urotensin-II does not play a major role in human CHF.     

Changes in gene expression involved in energy utilization during chicken follicle development

Ovarian follicle development in egg-laying species is characterized by rapid growth in 7 days prior to ovulation when DNA and protein synthesis is markedly increased in the granulosa and theca cells. However, energy and substrate sources to facilitate the extensive DNA and protein synthesis necessary for folliculogenesis have not been identified in avian species. The current study was undertaken to investigate the expression profiles of regulatory genes involved in glucose transport, glycolysis and fatty acid oxidation in the follicle membranes from the small white follicle (SWF) to follicle 1 (F1) stages of follicle development. In our analysis of glucose transporter (GLUT) isoform expression, the level of GLUT1 mRNA increased with follicle development while GLUT2, GLUT3 and GLUT8 mRNA levels were unaffected by follicle development. In contrast, the expression patterns of proteins involved in metabolism down-stream of glucose transport, including hexokinase (HK), pyruvate dehydrogenase E1alpha (PDH E1alpha) and citrate synthase (CS), did not vary with the developmental stage of the follicle, even during rapid follicle growth. Expression of genes related to beta-oxidation of fatty acids (carnitine palmityl CoA transferase I and II, l-3-hydroxyacyl CoA dehydrogenase and long-chain acyl-CoA dehydrogenase), for which expression in the ovarian follicles of mammalian species has not previously been studied, was not changed consistently with the follicle development. These results suggest that both glucose and fatty acids might work as energy sources to ensure rapid follicle development in the chicken ovary, even though glycolysis and beta-oxidation are not modulated by follicle development.