ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Inflammatory cells such as macrophages and T lymphocytes play an important role in vascular calcification associated with atherosclerosis and cardiac valvular disease. In particular, macrophages activated with cytokines derived from T lymphocytes such as interferon-&ggr; (IFN-&ggr;) may contribute to the development of vascular calcification. Moreover, we have shown the stimulatory effect of 1&agr;,25-dihydroxyvitamin D3(1,25(OH)2D3) on in vitro calcification through increasing the expression of alkaline phosphatase (ALP), an ectoenzyme indispensable for bone mineralization, in vascular smooth muscle cells. Therefore, we hypothesized that macrophages may induce calcifying phenotype, especially the expression of ALP in human vascular smooth muscle cells (HVSMCs) in the presence of IFN-&ggr; and 1,25(OH)2D3. To test this hypothesis, we used cocultures of HVSMCs with human monocytic cell line (THP-1) or peripheral blood monocytes (PBMCs) in the presence of IFN-&ggr; and 1,25(OH)2D3. THP-1 cells or PBMCs induced ALP activity and its gene expression in HVSMCs and the cells with high expression of ALP calcified their extracellular matrix by the addition of &bgr;-glycerophosphate. Thermostability and immunoassay showed that ALP induced in HVSMCs was bone-specific enzyme. We further identified tumor necrosis factor-&agr; (TNF-&agr;) and oncostatin M (OSM) as major factors inducing ALP in HVSMCs in the culture supernatants of THP-1 cells. TNF-&agr; and OSM, only when applied together, increased ALP activities and in vitro calcification in HVSMCs in the presence of IFN-&ggr; and 1,25(OH)2D3. These results suggest that macrophages may contribute to the development of vascular calcification through producing various inflammatory mediators, especially TNF-&agr; and OSM.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

The steroid hormone 1&agr;,25-dihydroxyvitamin D3[1&agr;, 25-(OH)2D3] promotes vascular smooth muscle cell (VSMC) growth and calcification, but the precise mechanism by which 1&agr;, 25-(OH)2D3regulates VSMC migration is unknown. In rat aortic SMCs, we found that 1&agr;, 25-(OH)2D3(0.1 to 100 nmol/L) induced a dose-dependent increase in VSMC migration. This response required the activation of phosphatidylinositol 3-kinase (PI3 kinase) because 1&agr;, 25-(OH)2D3-induced migration was completely abolished by the PI3 kinase inhibitors, LY294002 (10 &mgr;mol/L) or wortmannin (30 nmol/L). Furthermore, the RNA polymerase inhibitor, 5,6-dichlorobenzimidazole riboside (50 &mgr;mol/L), did not affect 1&agr;, 25-(OH)2D3-induced VSMC migration, suggesting that gene transcription is not involved in this rapid response. Using analogs of 1&agr;, 25-(OH)2D3, which have been characterized for their abilities to induce either transcriptional or nontranscriptional responses of 1&agr;, 25-(OH)2D3, we found that 1&agr;,25-dihydroxylumisterol, which is a potent agonist of the rapid, nongenomic responses, was equipotent with 1&agr;, 25-(OH)2D3in inducing PI3 kinase activity and VSMC migration. Moreover, 1&bgr;, 25-(OH)2D3, which specifically antagonizes the nongenomic actions of 1&agr;, 25-(OH)2D3, abolished 1&agr;, 25-(OH)2D3-induced PI3 kinase activity and VSMC migration, whereas the inhibitor of the genomic actions of vitamin D, (23S)-25-dehydro-1&agr;-OH-D3-26,23-lactone, did not affect these responses. These results indicate that 1&agr;, 25-(OH)2D3induces VSMC migration independent of gene transcription via PI3 kinase pathway, and suggest a possible mechanism by which 1&agr;, 25-(OH)2D3may contribute to neointima formation in atherosclerosis and vascular remodeling.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Interactions between integrins and growth factor receptors play a critical role in the development and healing of the vasculature. This study mapped two binding domains on fibronectin (FN) that modulate the activity of the angiogenic factor, vascular endothelial growth factor (VEGF). Using solid-phase assays and surface plasmon resonance analysis, we identified two novel VEGF binding domains within the N- and C-terminus of the FN molecule. Native FN bound to VEGF enhanced endothelial cell migration and mitogen-activated protein (MAP) kinase activity, but FN that is devoid of the VEGF binding domains failed to do so. Coprecipitation studies confirmed a direct physical association between VEGF receptor-2 (Flk-1) and the FN integrin, &agr;5&bgr;1, which required intact FN because FN fragments lacking the VEGF binding domains failed to support receptor association. Thrombin-activated platelets released intact VEGF/FN complexes, which stimulated endothelial cell migration and could be inhibited by soluble high affinity VEGF receptor 1 and antibodies to &agr;5&bgr;1integrin. This study demonstrates that FN is potentially a physiological cofactor for VEGF and provides insights into mechanisms by which growth factor receptors and integrins cooperate to influence cellular behavior.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

We have previously demonstrated that growth of embryonic chick atrial cells in medium supplemented with lipoprotein-depleted serum (LPDS) resulted in a coordinate increase in the expression of genes involved in the parasympathetic response of the heart (the M2muscarinic receptor; the &agr;-subunit of the heterotrimeric G protein, G&agr;i2; and the inward rectifying K+channel protein, GIRK1) and a marked increase in the negative chronotropic response of atrial cells to muscarinic stimulation. In the present study, we demonstrate that regulation of G&agr;i2promoter activity by LPDS is mediated by the binding of a sterol regulatory element binding protein (SREBP) to a sterol regulatory element (SRE) in the G&agr;i2promoter. Deletion and point mutation of this putative SRE interfered with the regulation of the G&agr;i2promoter by SREBP and LPDS. Furthermore gel shift assays demonstrated that point mutations in the putative G&agr;i2SRE markedly inhibited the binding of purified SREBP to oligonucleotides containing the G&agr;i2SRE sequence. The expression of a dominant-negative SREBP mutant interfered with LPDS stimulation of G&agr;i2promoter activity. Finally, we demonstrate that SREBP-1 is markedly more potent than SREBP-2 for the stimulation of G&agr;i2promoter activity, suggesting that SREBP1 may play a role in the regulation of G&agr;i2expression. These are the first data to demonstrate SREBP regulation of a protein not involved in lipid homeostasis and suggest a new relationship between lipid metabolism and the parasympathetic response of the heart.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Tyrosine kinase cascades may play a role in the hypoxic regulation of hypoxia-inducible factor (HIF)-1. We investigated the role of tyrosine kinase phosphorylation and of the Shc/Ras cascade on hypoxic HIF-1 stabilization. Exposure of human umbilical vein endothelial cells to hypoxia results in HIF protein stabilization as early as 10 minutes, with a maximum at 3 hours, and also in Shc tyrosine phosphorylation, with a maximum at 10 minutes. To test whether Shc directly mediates hypoxia-induced HIF stabilization, human umbilical vein endothelial cells were transfected with a dominant-negative Shc mutant (dnShc), resulting in significantly reduced HIF protein levels compared with control. Similar results were obtained with cells transfected with dominant-negative Ras, a known downstream effector of Shc. Hypoxia-induced Ras activity was significantly reduced in cells transfected with dnShc compared with control levels, indicating that Ras indeed acts downstream from Shc. Moreover, cells pretreated with a specific Raf-1 kinase inhibitor, a known downstream effector of Ras, exhibited reduced HIF protein levels. To examine the functional consequences of Shc in hypoxic signaling, HIF-1 ubiquitination, protein stabilization, and endothelial cell migration were assessed. Overexpression of dnShc increased ubiquitination of HIF-1 and reduced the half-life of the protein. Moreover, dnShc, dominant-negative Ras, or the Raf-1 kinase inhibitor significantly inhibited migration under hypoxia. Thus, Shc in concert with Ras and Raf-1 contributes to hypoxia-induced HIF-1&agr; protein stabilization and endothelial cell migration.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Compartmentation of intracellular signaling pathways serves as an important mechanism conferring the specificity of G protein-coupled receptor (GPCR) signaling. In the heart, stimulation of &bgr;2-adrenoceptor (&bgr;2-AR), a prototypical GPCR, activates a tightly localized protein kinase A (PKA) signaling, which regulates substrates at cell surface membranes, bypassing cytosolic target proteins (eg, phospholamban). Although a concurrent activation of &bgr;2-AR-coupled Giproteins has been implicated in the functional compartmentation of PKA signaling, the exact mechanism underlying the restriction of the &bgr;2-AR-PKA pathway remains unclear. In the present study, we demonstrate that phosphatidylinositol 3-kinase (PI3K) plays an essential role in confining the &bgr;2-AR-PKA signaling. Inhibition of PI3K with LY294002 or wortmannin enables &bgr;2-AR-PKA signaling to reach intracellular substrates, as manifested by a robust increase in phosphorylation of phospholamban, and markedly enhances the receptor-mediated positive contractile and relaxant responses in cardiac myocytes. These potentiating effects of PI3K inhibitors are not accompanied by an increase in &bgr;2-AR-induced cAMP formation. Blocking Gior G&bgr;&ggr; signaling with pertussis toxin or &bgr;ARK-ct, a peptide inhibitor of G&bgr;&ggr;, completely prevents the potentiating effects induced by PI3K inhibition, indicating that the pathway responsible for the functional compartmentation of &bgr;2-AR-PKA signaling sequentially involves Gi, G&bgr;&ggr;, and PI3K. Thus, PI3K constitutes a key downstream event of &bgr;2-AR-Gisignaling, which confines and negates the concurrent &bgr;2-AR/Gs-mediated PKA signaling.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID