Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells and promotes neovascularization in vivo. To determine whether interleukin-1 beta (IL-1 beta), which is present in atherosclerotic lesions, induces VEGF gene expression in vascular smooth muscle cells, we performed RNA blot analysis on rat aortic smooth muscle cells (RASMC) with a rat VEGF cDNA probe. IL-1 beta increased VEGF mRNA levels in RASMC in a time- and dose-dependent manner. As little as 0.1 ng/ml IL-1 beta increased VEGF mRNA levels by 2-fold and 10 ng/ml IL-1 beta increased VEGF mRNA by 4-fold. We also measured the half-life of VEGF mRNA and performed nuclear run-on experiments before and after addition of IL-1 beta to see if IL-1 beta increased VEGF mRNA levels by stabilizing the mRNA or by increasing its rate of transcription. The normal, 2-h half-life of VEGF mRNA in RASMC was lengthened to 3.2 h (60%) by IL-1 beta, and IL-1 beta increased the rate of VEGF gene transcription by 2.1-fold. In immunoblot experiments with an antibody specific for VEGF, we found that IL-1 beta increased VEGF protein levels in RASMC by 3.3-fold. Together these data indicate that IL-1 beta induces VEGF gene expression in smooth muscle cells. This IL-1 beta-induced expression of VEGF may accelerate the progression of atherosclerotic lesions by promoting the development of new blood vessels.

No abstract

Contact inhibition mediates monolayer formation and withdrawal from the cell cycle in vascular endothelial cells. In studying the cyclins-key regulators of the cell cycle-in bovine aortic endothelial cells (BAEC), we found that levels of cyclin A mRNA decreased in confluent BAEC despite the presence of 10% fetal calf serum. We then transfected into BAEC a series of plasmids containing various lengths of the human cyclin A 5 flanking sequence and the luciferase gene. Plasmids containing 3,200, 516, 406, 266, or 133 bp of the human cyclin A promoter directed high luciferase activity in growing but not confluent BAEC. In contrast, a plasmid containing 23 bp of the cyclin A promoter was associated with a 65-fold reduction in activity in growing BAEC, and the promoter activities of this plasmid were identical in both growing and confluent BAEC. Mutation of the activating transcription factor (ATF) consensus sequence at bp ؊80 to ؊73 of the cyclin A promoter decreased its activity, indicating the critical role of the ATF site. We identified by gel mobility shift analysis protein complexes that bound to the ATF site in nuclear extracts from growing but not confluent BAEC and identified (with antibodies) ATF-1 as a binding protein in nuclear extracts from growing cells. Also, ATF-1 mRNA levels decreased in confluent BAEC. Taken together, these data suggest that the ATF site and its cognate binding proteins play an important role in the downregulation of cyclin A gene expression during contact inhibition.The endothelial cell layer is important in thrombosis, thrombolysis, lymphocyte homing, inflammation, modulation of the immune response, and regulation of vascular tone. Growth inhibition and cell cycle withdrawal mediated by contact, characteristic of most normal cell types, are critical for monolayer formation by vascular endothelial cells both in vivo and in vitro. Yet despite extensive research, little is known about the molecular mechanisms of contact inhibition in these cells (2,17,29).The progress of the cell cycle is regulated by the sequential expression of cyclins and the activation of their associated cyclin-dependent kinases (Cdks) (22, 43). p27, a recently identified inhibitor of Cdk2 and Cdk4, is activated in contactinhibited cells (36,38,39,45). Therefore, the activation of Cdk inhibitors such as p27 may explain in part the process of contact inhibition. It is just as likely, however, that contact inhibition results from downregulation of Cdk activators like the cyclins. Indeed, the cyclin A gene has been identified by differential screening as a gene downregulated at confluence in Mv1Lu cells (40).Cyclin A associates with Cdk2 in the S phase of the cell cycle and with Cdc2 in the G 2 /M phase, and it is required for DNA replication in the S phase (6,13,32,37,43,44). Cyclin A can also affect G 1 /S transit, as overexpression of cyclin A overcomes the G 1 /S block induced by loss of cell adhesion (16). The genomic organization of the human cyclin A gene was published recently (20,50). The gene contains eigh...

No abstract

Lycium barbarum is a boxthorn that produces the goji berries. The aim of the current study was to evaluate the proliferative effect of L. barbarum polysaccharides (LBP) on probiotics. LBP was extracted from goji berries and its monosaccharide composition characterized by gas chromatography (GC). The LBP extract contained arabinose, rhamnose, xylose, mannose, galactose, and glucose. LBP obviously promoted the proliferation of lactic acid bacteria (LAB) strains, especially Bifidobacterium longum subsp. infantis Bi-26 and Lactobacillus acidophilus NCFM. In the presence of LBP in the growth medium, the β-galactosidase (β-GAL) and lactate dehydrogenase (LDH) activities of strain Bi-26 significantly increased. The activities of β-GAL, LDH, hexokinase (HK), 6-phosphofructokinase (PFK), and pyruvate kinase (PK) of strain NCFM significantly increased under those conditions. LAB transcriptome sequencing analysis was performed to elucidate the mechanism responsible for the proliferative effect of LBP. The data revealed that LBP promoted the bacterial biosynthetic and metabolic processes, gene expression, transcription, and transmembrane transport. Pyruvate metabolism, carbon metabolism, phosphotransferase system (PTS), and glycolysis/gluconeogenesis genes were overexpressed. Furthermore, LBP improved cell vitality during freeze-drying and tolerance of the gastrointestinal environment. In summary, LBP can be used as a potential prebiotic for Bifidobacterium and Lactobacillus.