PubMed · 13571629
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R DE BELLO, O ETORENA, R RODRIGUEZ BARRIOS. [Pulseless disease].. https://pubmed.ncbi.nlm.nih.gov/13571629/
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We have utilized serial analysis of gene expression (SAGE) to analyze the temporal response of human aortic endothelial cells (HAECs) to short-term chronic hypoxia at the level of transcription. Primary cultures of HAECs were exposed to 1% O2 hypoxia for 8 and 24 h and compared with identical same passage cells cultured under standard (5% CO2-95% air) conditions. A total of 121,446 tags representing 37,096 unique tags were sequenced and genes whose expression levels were modulated by hypoxia identified by novel statistical analyses. Hierarchical clustering of genes displaying statistically significant hypoxia-responsive alterations in expression revealed temporal modulation of a number of major functional gene families including those encoding heat shock factors, glycolytic enzymes, extracellular matrix factors, cytoskeletal factors, apoptotic factors, cell cycle regulators and angiogenic factors. Within these families we documented the coordinated modulation of both previously known hypoxia-responsive genes, numerous genes whose expressions have not been previously shown to be altered by hypoxia, tags matching uncharacterized UniGene entries and entirely novel tags with no UniGene match. These preliminary data, which indicate a reduction in cell cycle progression, elevated metabolic stress and increased cytoskeletal remodeling under acute hypoxic stress, provide a foundation for further analyses of the molecular mechanisms underlying the endothelial response to short-term chronic hypoxia.
Smooth muscle cell (SMC) proliferation and migration are key processes that occur in the pathogenesis of atherosclerosis and post-angioplasty restenosis. In the present study, we designed locked nucleic acid (LNA)-modified DNAzymes targeting a specific region spanning the translational start site of human EGR-1, an immediate-early gene, wherein two of the nucleotides in each of the 9+9 hybridizing arms of the DNAzyme were substituted with LNA monomers. In vitro cleavage experiments revealed that the LNA- modified DNAzyme (LzF4) cleaved a 32P-labelled 388 nt EGR-1 transcript with greater efficacy than its native unmodified phosphodiester counterpart, DzF. The scrambled versions of these molecules, LzF4SCR and DzFSCR, did not display any ability to cleave the transcript. Western blot analysis revealed that both active molecules abrogated serum-inducible EGR-1 protein expression in primary human aortic SMCs and inhibited serum-inducible SMC proliferation in a dose-dependent and non-toxic manner. SMC proliferation was inhibited by >50% with LzF4 at concentrations as low as 20 nM, whereas inhibition by DzF at this concentration was not evident. Finally, LzF4 and DzF inhibited SMC regrowth from the wound edge after mechanical injury in vitro. In contrast, neither DzFSCR nor LzF4SCR had any influence on EGR-1 protein expression, SMC proliferation or regrowth. These findings provide the first functional demonstration of LNA-modified DNAzyme efficacy in a biological setting of any kind. These studies also demonstrate that LNA modification increases DNAzyme potency without necessarily compromising specificity.
BACKGROUND AND OBJECTIVES: Endothelial and monocytic cells appear to play a key role in the initiation and progression of atherosclerosis and restenosis via the upregulation of inflammatory cytokines and the formation of oxidized low-density lipoprotein (ox-LDL). However, the role of smooth muscle cells (SMCs) has been underestimated and is not well understood. It was investigated for the first time that native LDL stimulates human SMCs to secrete IL-8. The aim of this study was to investigate the signaling pathway involved in the upregulation of IL-8, induced by LDL in human aortic SMCs. METHODS AND RESULTS: LDL-induced IL-8 expression (mRNA and protein) is specific to SMCs and likely to be regulated at the transcription level in dose- and time-dependent manners, as judged by experiments with actinomycin D and ELISA. Although both p38 and ERK 1/2 MAPKs were activated by LDL, only p38 MAPK is responsible for the LDL effects, as evidenced by a complete blockade of IL-8 upregulation by SB203580. Pretreatment with catalase significantly decreased the extent of IL-8 upregulation, indicating that H2O2 is necessary for the LDL response. Activation of activator protein (AP)-1, but not nuclear factor (NF)-kappaB, by p38 or H2O2 appears to be necessary along with the concomitant upregulation of c-fos and c-JUN, as judged by electrophoretic mobility shift and luciferase reporter assays. CONCLUSIONS: These data demonstrated that LDL stimulates SMCs to induce IL-8 production in dose- and time-dependent manners at the transcription level and that the LDL signaling in hAoSMCs is conveyed via the generation of H2O2, the phosphorylation of p38 MAPK, the activation of AP-1, and the participation of NF-kappaB.